基于胶体金标记米曲霉素的免疫层析试纸法快速筛查SLE

建立基于胶体金标记米曲霉素(AOL)的快速筛查系统性红斑狼疮(SLE)的免疫层析试纸法。

利用大肠杆菌BL21原核表达特异性识别核心岩藻糖基的AOL基因(FleA), 并进行纯化及胶体金标记。利用特异性识别IgG的Protein G以及胶体金标记的AOL共同建立快速筛查SLE的免疫层析试纸法(ICS)。

成功表达并纯化AOL重组蛋白, 并进行胶体金标记; 应用胶体金标记AOL的ICS检测SLE患者血清呈阳性反应, 而健康对照及其他自身免疫性疾病(类风湿关节炎、IgA肾病和结缔组织病等)呈阴性反应。

成功建立基于胶体金标记AOL的快速筛查SLE的ICS, 为SLE早期筛查提供了可靠依据。

     

2015-2020年大连市流感病毒分离鉴定情况分析

对2015-2020年大连市流感病毒分离鉴定情况进行对比分析, 为大连市流感防控工作提供参考。

采集大连市2家国家级流感监测哨点医院的流感样病例咽拭子样本, 用MDCK细胞和鸡胚分别进行病毒分离培养, 并采用HA试验和HI试验对分离的病毒滴度和型别进行鉴定。

2015-2020年共分离培养流感病毒核酸检测阳性的咽拭子1 055份, 其中MDCK细胞分离出流感病毒501株, 鸡胚分离出流感病毒72株, 总体病毒分离率54.31%。MDCK细胞分离出A(H1N1)、A(H3N2)、B(Victoria)和B(Yamagata)型病毒, 鸡胚对A(H3N2)型病毒不敏感, 但可以分离出A(H1N1)、B(Victoria)和B(Yamagata)型病毒。每年的优势毒株虽不同, 但分离流感病毒的月份均在流行季内, 与北方流行形势一致。

MDCK细胞与鸡胚的流感病毒分离率不同。大连市每年流感流行的优势株和流行程度虽不同, 但流行程度处于相对平稳状态。

     

腹泻仔猪大肠埃希菌分离株ST4214的全基因组测序分析

分析腹泻仔猪大肠埃希菌(E.coli)分离株血清型、毒力因子及耐药基因。

本课题组前期研究从腹泻仔猪粪便样本中分离到64株E.coli, 本研究取其中含有毒力因子最多的1株进行全基因组测序分析。

E.coli分离株基因组序列为5.5 Mb, 预测蛋白质编码序列数为5 743, 与已知致病菌E.coli O145的基因重合率较低, 为64.53%。经多位点序列分型分析, 确定分离株为E.coli ST4214, 血清型为O3∶H45。与病原菌O157∶H7、O145∶H28和O104∶H4的毒力基因比较表明: 4株菌共同含有的毒力基因为354个, E.coli ST4214含有64种独特的毒力基因、编码鞭毛蛋白、Ⅳ型分泌蛋白、Ⅱ型分泌蛋白、溶血素、菌毛、肠毒素、荚膜多糖相关蛋白质。耐药基因E.coli分离株含有氨基香豆素抗性基因、磺胺类耐药基因、β-内酰胺抗性基因、多粘菌素耐药基因、肽抗生素抗性基因和抗生素耐药基因外排泵等多重耐药基因。

腹泻仔猪E.coli ST4214分离株是一株新菌株, 具有较多毒力因子及耐药基因, 可能与仔猪腹泻发病相关。

     

卡介苗荚膜阿拉伯甘露聚糖的提纯鉴定及其免疫调节功能的初步研究

确定卡介苗(BCG)中荚膜中糖成分阿拉伯甘露聚糖(AM)的功能。

本研究从我国目前普遍使用的BCG中, 利用氯仿甲醇抽提方法分离纯化荚膜AM, 经AM单克隆抗体鉴定后, 腹腔注射BCG致敏小鼠, 体内外评价AM的免疫调节功能。

AM能在小鼠体内增强BCG诱导的迟发型超敏反应(P < 0.01), 体外免疫指标检测发现AM提高小鼠脾细胞Th1类细胞因子IFN-γ和TNF-α的表达水平(P < 0.05)。

BCG的荚膜AM是潜在的保护性抗原, 并为优化BCG提供理论依据。

     

耐碳青霉烯类鲍曼不动杆菌同源性及生物膜形成能力分析

分析耐碳青霉烯类鲍曼不动杆菌(Carbapenem Resistant Acinetobacter baumannii,CRAB)的同源性,测定其生物膜形成能力,分析blaOXA基因携带与生物膜形成能力的相关性,探讨CRAB在医院暴发的影响因素。收集佳木斯大学附属第一医院非重复CRAB共35株,全自动微生物鉴定系统VITEK-II及体外敏感实验筛选CRAB,多序列位点分型(MLST)、肠道细菌基因重复序列聚合酶链式反应(ERIC-PCR)分析其同源性,结晶紫染色法定量检测生物膜形成能力。33株CRAB为ST 2型,1株ST 671型,1株ST 1199型为新发现的MLST分型。ERIC可分为A、B、C、D四型,A型9株,B型24株,C型1株,D型1株。CRAB及碳青霉烯类敏感鲍曼不动杆菌(CSAB)组生物膜形成能力差异有统计学意义(0.330±0.258、0.534±0.402,P0.05);blaOXA-23、blaOXA-51、blaOXA-24基因携带与CRAB生物膜形成能力无明确相关性(P0.05)。生物膜形成能力与碳青霉烯类耐药性的获得呈现负相关。ERIC-PCR分辨率较强,且与MLST具有一定相关性。ST2型为主要流行菌株,患者的高危因素以及医护人员的消毒意识可能与CRAB的暴发有关。     

辽宁省食源性副溶血性弧菌毒力基因、血清分型及耐药性分析

研究辽宁省2017-2020年市售水产品及其制品中分离的158株副溶血性弧菌的毒力基因、血清分型和耐药性, 为食源性疾病的暴发和散发进行评估和预警。

采用多重荧光定量PCR技术对毒力基因tlh、tdh和trh进行检测, 同时检测血清分型; 采用肉汤稀释法测定副溶血性弧菌MIC值并分析其耐药性。

158株副溶血性弧菌中均携带tlh基因, 携带tdh基因的菌株2株, 携带trh基因的菌株2株, 同时携带tdh基因和trh基因的菌株1株; 158株中67株血清型为O2(含K不分型), 39株为O3(含K不分型), 携带tdh和trh毒力基因的菌株血清型均为O3;副溶血性弧菌122株(77.22%)对头孢唑啉耐药, 36株(22.78%)对头孢西丁耐药, 30株(18.99%)对氨苄西林耐药, 18株菌呈现耐受2类及以上的多重耐药性。携带毒力基因的5株副溶血性弧菌对头孢唑啉均耐药。

大多数食源性副溶血性弧菌不携带毒力基因, O2血清型(42.41%)是辽宁省食源性副溶血性弧菌的主要血清型, 其次为O3(24.68%); 副溶血性弧菌对头孢唑啉显著耐药, 少数菌表现出多重耐药, 提示应继续加强水产品及其生食制品中副溶血性弧菌的致病性和耐药性监测。

     

云南兰坪铅锌尾矿区可培养细菌的分离及其酶活筛选

对云南兰坪铅锌尾矿区样品中的可培养细菌进行分离及初步鉴定, 同时挖掘具有酶活性功能的菌株。

从兰坪铅锌尾矿区及周边农田采集了20份样品, 运用10种培养基进行细菌分离, 对分离菌株的16S rRNA基因测序以鉴定其分类地位, 再以平板透明圈法检测分离菌株的淀粉酶、纤维素酶、蛋白酶和脂肪酶活性。

从20份样品中分离获得了320株细菌, 隶属于6个纲、14个目、26个科、39个属, 有5个潜在新分类单元。其中链霉菌属的菌株数最多, 高达102株, 占菌株总数的31.88%, 为优势种群; 其次为芽胞杆菌属菌株40株, 肠杆菌属菌株17株。去重后对165株菌进行酶活检测, 有41株菌具有淀粉酶活性, 占筛选菌株总数的24.70%, 主要为链霉菌属和芽胞杆菌属; 有40株菌对纤维素酶具有活性, 占筛选菌株总数的24.10%, 主要为链霉菌属和芽胞杆菌属; 有14株菌对蛋白酶具有活性, 占筛选菌株总数的8.43%, 主要为链霉菌属和芽胞杆菌属; 有12株菌对脂肪酶具有活性, 占筛选菌株总数的7.23%, 主要为链假单胞菌属。

兰坪铅锌尾矿区可培养细菌的种类丰富, 且蕴藏着大量具有酶活性的菌株。研究结果为了解兰坪铅锌尾矿细菌多样性提供了数据参考, 同时也为酶工业的研究开发提供了更多的菌种资源。

     

基于Web of Science的阴道菌群研究现状和热点的可视化分析与展望

探索阴道菌群近10年来的研究现状并预测其发展趋势。

在Web of Science数据库中检索近10年阴道菌群相关的文献,运用可视化软件VOSviewer进行共词分析、文献耦合、合著分析等。

阴道菌群领域发文量呈整体上升趋势,目前的主要热点研究分为阴道菌群的多样性、传染病学、细胞生物和基因学、阴道菌群与生殖的关联四大方面。

文献可视化研究展现了阴道菌群的研究现状和发展趋势,为相关研究人员提供了借鉴。

     

杀菌型副干酪乳酪杆菌N1115发酵乳饮品对小鼠免疫功能的影响

研究杀菌型副干酪乳酪杆菌N1115（Lacticaseibacillus paracasei N1115）发酵乳饮品对小鼠免疫脏器指数、免疫球蛋白、巨噬细胞、NK细胞、B淋巴细胞及T淋巴细胞的影响。

将SPF级6～8周龄雄性Balb/c小鼠随机分为对照组以及杀菌型副干酪乳酪杆菌N1115发酵乳饮品低、中、高剂量组,每组15只,连续灌胃30 d,进行免疫脏器指数测定、免疫球蛋白测定、碳廓清能力测定、腹腔巨噬细胞吞噬鸡红细胞实验、NK细胞活性测定、脾淋巴细胞转化实验、迟发型变态反应、血清溶血素测定和抗体细胞生成实验。

杀菌型副干酪乳酪杆菌N1115发酵乳饮品低、中、高剂量组的小鼠碳廓清指数显著高于对照组（t = 3.926 2,P = 0.000 7;t = 6.000 1,P＜0.000 1;t = 5.314 4,P＜0.000 1）,腹腔巨噬细胞吞噬鸡红细胞的吞噬率显著高于对照组（t = 3.812 1,P = 0.001 5;t = 4.257 2,P = 0.000 4;t = 4.976 3,P = 0.000 5）。

杀菌型副干酪乳酪杆菌N1115发酵乳饮品具有增强小鼠非特异性免疫力的功能,可为副干酪乳酪杆菌N1115的开发利用提供科学依据。

     

哮喘患儿口咽部菌群分析及3株优势菌的拮抗作用

了解哮喘患儿呼吸道菌群多样性及其组成特征, 同时研究所分离的3株优势菌对流感嗜血杆菌的抑制作用, 探究哮喘与呼吸道菌群之间的关系。

采集沈阳市儿童医院呼吸内科2019年3月至2019年12月收治的21例4~12岁急性发作期哮喘患儿咽拭标本, 并同时采集23例同龄健康儿童的咽拭标本作为对照, 对呼吸道菌群进行分离培养、纯化和16S rRNA鉴定。采用牛津杯法检测健康儿童口咽部分离的3株优势菌对流感嗜血杆菌的拮抗作用。

哮喘和健康儿童呼吸道培养出的需氧菌(t=2.143, P=0.038)和厌氧菌(t=3.270, P=0.002)的密度差异有统计学意义。哮喘患儿咽部需氧菌以肺炎链球菌和流感嗜血杆菌为主, 厌氧菌以韦荣球菌为主。健康儿童咽部需氧菌以缓症链球菌和口腔链球菌为主, 厌氧菌以干酪乳杆菌为主。健康儿童口咽部的3株优势菌对哮喘儿童口咽部流感嗜血杆菌的生长具有显著抑制作用。

与健康儿童相比, 哮喘患儿口咽部菌群发生紊乱, 且哮喘患儿口咽部需氧菌、厌氧菌密度显著增加。健康儿童口咽部的某些优势菌可能对哮喘致病菌的定植有一定的拮抗作用。

     

Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST)

ABSTRACT: BACKGROUND: In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis. RESULTS: Defined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering. CONCLUSIONS: MALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii, L. interrogans and L. kirschneri revealed distinctive, reproducible differentiating peaks for seven leptospiral strains which represent seven serovars. Results obtained by MALDI-TOF MS were confirmed by MLST and 16S rRNA gene sequencing.     

MALDI-TOF MS对肺炎链球菌鉴定与分型的应用

目的 探讨MALDI-TOF MS对肺炎链球菌鉴定和质谱分型的应用价值。方法 收集2009年1月至2013年5月温州医科大学附属第二医院临床分离的112株肺炎链球菌标本,采用Optochin敏感试验和全自动细菌分析仪对收集的菌株进行鉴定验证,并用Microflex MALDI-TOF质谱仪进行分析鉴定。根据质谱图的相似性进行细菌同源聚类树分析并构建质谱分型模型,采用荚膜肿胀试验对参与分型的菌株进行血清型比较。结果 除20株不符合检测条件之外,92株临床菌株和1株标准株经质谱分析均为肺炎链球菌,选取的60株菌株以0.5的差异水平,将60株肺炎链球菌分为18个质谱型别,在这些菌株的血清分型中有19F、19A、23F、23A、3和14六个血清型别,分布于不同的MALDI-TOF MS分型中,其中19F有18株,占30%（18/60）,分布在6种不同的MALDI-TOF MS分型中,也有3型血清型较为集中地分布于相应的MALDI-TOF MS一个型别里。结论 MALDI-TOF MS能快速、准确、简便地鉴定肺炎链球菌,且能达到种的水平。对比血清型,按照0.5差异水平,建立的18个质谱分型部分的型别与血清型有一致性,但也存有差异。     

利用基因组学和MALDI-TOF MS技术鉴定放线菌纲细菌的核糖体蛋白质标志物

【目的】基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)法基于微生物的特征蛋白指纹图谱鉴定菌种,本研究利用基因组学和MALDI-TOFMS技术鉴定放线菌纲细菌的核糖体蛋白质标志物。【方法】从MALDI-TOF MS图谱数据库选取放线菌纲代表菌种,在基因组数据库检索目标菌种,获取目标菌株或其参比菌株的核糖体蛋白质序列,计算获得分子质量理论值,用于注释目标菌株MALDI-TOFMS指纹图谱中的核糖体蛋白质信号。【结果】从8目,24科,53属,114种,142株放线菌的MALDI-TOFMS图谱中总共注释出31种核糖体蛋白质。各菌株的指纹图谱中核糖体蛋白质信号数量差异显著。各种核糖体蛋白质信号的注释次数差异显著。总共15种核糖体蛋白质在超过半数图谱中得到注释,注释次数最高的是核糖体大亚基蛋白质L36。【结论】本研究找到了放线菌纲细菌MALDI-TOF MS图谱中常见的15种核糖体蛋白质信号,可为通过识别核糖体蛋白质的质谱特征峰鉴定放线菌的方法建立提供依据。     

Difference and consensus of whole cell Salmonella enterica subsp. enterica serovars matrix-assisted laser desorption/ionization time-of-flight mass spectrometry spectra

AIM: Application of MALDI-TOF MS for characterization of strains of Salmonella enterica subsp. enterica. METHODS AND RESULTS: Whole cells were analysed by MALDI-TOF MS. Spectra with a maximum of 500 mass peaks between (m/z) 0 and 25000 were examined for consensus peaks manually and by a computer software algorithm. Consensus peaks were observed by both methods for spectra of Salmonella enterica serovars Derby, Hadar, Virchow, Anatum, Typhimurium and Enteritidis. CONCLUSIONS: Differences in numbers of consensus peaks in spectra obtained by manual and computer comparison indicated that development of the software involving statistical analysis of peak accuracy is necessary. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of an analysis system for peak profiles in whole cell MALDI-TOF MS spectra to enable intra and interlaboratory comparison.     

Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid identification of bacteria cultured in liquid media

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used for many years to rapidly identify whole bacteria. However, no consistent methodology exists for the rapid identification of bacteria cultured in liquid media. Thus, in this study we explored the use of MALDI-TOF MS analysis for rapid identification of cells cultured in liquid media. We determined that 2,5-dihydroxybenzoic acid (50 mg mL^?1, 50% acetonitrile, 0.1% trifluoroacetic acid) was the best matrix solution for MALDI-TOF MS for this type of study. Moreover, the tested strains were successfully differentiated by principal component analysis, and the main characteristics of the mass peaks for each species were found in mixed culture samples. In addition, we found that the minimum number of cells for detection was 1.8×10³. In conclusion, our findings suggest that MS-based techniques can be developed as an auxiliary method for rapidly and accurately identifying bacteria cultured in liquid media.     

Rapid detection of carbapenem resistance in Acinetobacter baumannii using matrix-assisted laser desorption ionization-time of flight mass spectrometry

Rapid detection of carbapenem-resistant Acinetobacter baumannii strains is critical and will benefit patient care by optimizing antibiotic therapies and preventing outbreaks. Herein we describe the development and successful application of a mass spectrometry profile generated by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) that utilized the imipenem antibiotic for the detection of carbapenem resistance in a large series of A. baumannii clinical isolates from France and Algeria. A total of 106 A. baumannii strains including 63 well-characterized carbapenemase-producing and 43 non-carbapenemase-producing strains, as well as 43 control strains (7 carbapenem-resistant and 36 carbapenem-sensitive strains) were studied. After an incubation of bacteria with imipenem for up to 4 h, the mixture was centrifuged and the supernatant analyzed by MALDI-TOF MS. The presence and absence of peaks representing imipenem and its natural metabolite was analyzed. The result was interpreted as positive for carbapenemase production if the specific peak for imipenem at 300.0 m/z disappeared during the incubation time and if the peak of the natural metabolite at 254.0 m/z increased as measured by the area under the curves leading to a ratio between the peak for imipenem and its metabolite being <0.5. This assay, which was applied to the large series of A. baumannii clinical isolates, showed a sensitivity of 100.0% and a specificity of 100.0%. Our study is the first to demonstrate that this quick and simple assay can be used as a routine tool as a point-of-care method for the identification of A. baumannii carbapenemase-producers in an effort to prevent outbreaks and the spread of uncontrollable superbugs.     

MALDI-TOF MS与16S rDNA方法对弧菌科微生物的鉴定与系统分类学分析

目的 比较基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）与16S rDNA方法对弧菌科微生物的鉴定及系统分类学分析能力。方法 对19株弧菌科微生物,采用MALDI-TOF MS进行蛋白质图谱采集,通过对特征峰的分析,实现对微生物的鉴定和系统分类学分析;同时对19株微生物进行16S rDNA测序,用邻接法对16S rDNA序列进行鉴定和系统分类学分析,比较两种方法在弧菌科微生物鉴定和系统分类学分析中的异同。结果 两种方法对19株弧菌科微生物的种属鉴定结果一致。系统分类学分析中,多株同种属的弧菌的两种方法分析结果一致,但对拟态弧菌和霍乱弧菌在树状图中的位置和亲缘关系,两种方法差异较大。结论 MALDI-TOF MS与16S rDNA均能够快速准确地鉴定弧菌科微生物,但利用MALDI-TOF MS进行系统分类学分析还有待数据库的扩大及算法的优化。     

Characterization of ��/��- and ��-Gliadins in Commercial Varieties and Breeding Lines of Durum Wheat Using MALDI-TOF and A-PAGE Gels

In this work, gliadin composition has been analyzed in 33 accessions of durum wheat using MALDI-TOF MS and compared with A-PAGE results. The MALDI-TOF MS spectra were 29,900-42,500 Da, which corresponds to the α/β- and γ-gliadin regions in A-PAGE. The average of gliadin peaks per line was 23 for MALDI-TOF MS and only 14.8 bands for A-PAGE. MALDI-TOF MS identified 33 gliadin peaks in the durum wheat collection, 20 of which were unique peaks present in 7 lines. A-PAGE analysis identified 30 bands, of which only 4 were unique. Thus, the MALDI-TOF MS method was more sensitive than A-PAGE for identifying α/β- and γ-gliadins in the 33 durum wheat lines studied. Phylogenetic analyses performed using MALDI-TOF MS data assigned the durum wheat lines to two groups. The utility of MALDI-TOF MS to determine relationships among genotypes and for identification of durum wheat accessions is discussed.     

MHT和mCIM试验检测肠杆菌科金属碳青霉烯酶效能的评价

目的 探讨改良Hodge试验（MHT）及改良碳青霉烯灭活试验（mCIM）检测肠杆菌科细菌金属碳青霉烯酶的应用价值。方法 VITEK 2 Compact全自动细菌鉴定及药敏系统进行细菌鉴定和药敏试验,筛选2015-2017年非重复临床分离的碳青霉烯类耐药的肠杆菌科细菌,MHT及mCIM进行产碳青霉烯酶表型确证试验,PCR检测常见的金属碳青霉烯酶IMP-4、IMP-8、VIM-1、VIM-2、NDM基因。比较MHT及mCIM对肠杆菌科金属碳青霉烯酶的检测效能。结果 本实验共收集40株临床分离菌株,MHT阳性36株,阳性率90.0%。mCIM阳性39株,阳性率为97.5%。PCR产物测序Blast比对证实4株为产IMP酶菌株,5株产NDM型菌株,未检测到VIM基因。MHT试验检测IMP酶的灵敏度、特异性、阳性预测值、阴性预测值分别为100.0%、11.1%、11.1%、100.0%,MHT检测NDM酶分别为40.0%、2.9%、5.6%、25.0%。mCIM检测IMP酶的灵敏度、特异性、阳性预测值、阴性预测值分别100.0%、2.8%、10.3%、100.0%,mCIM检测NDM型分别为100.0%、2.9%、12.8%、100.0%。结论 MHT及mCIM检测肠杆菌科细菌产金属碳青霉烯酶具有良好的灵敏度,但特异性偏低,应结合分子生物学方法进行检测,为感染控制提供保障。     

Specific peptide patterns of follicular fluids at different growth stages analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Human follicular fluid (HFF) has been suggested to influence oocyte development potential, and some of HFF proteins may be potential markers for oocyte maturation during follicular development. Using matrix-assisted laser desorption /ionization-time of flight mass spectrometry (MALDI-TOF MS), the presence of specific peptide peaks in HFF which could represent the follicle development potential was evaluated. HFF from different developmental stages were first digested and the resultant peptide mixtures were directly analyzed by MALDI-TOF MS. It was shown that the frequencies of specific peaks demonstrated higher reproducibility than peak intensities after multiple measurements (>or=6 times) per sample. Using this approach, a reliable peak list for each different sample could be generated by combining the information from multiple measurements. By comparing the peak lists from different samples at different growth stages, we found that 5 specific peaks appeared in the 100% frequency category of 6 replicates in all the HFF samples containing mature oocyte. Similarly, such 25 peptide peaks were also identified for HFF containing immature oocyte. These specific peaks could be used to distinguish HFF from different stages as biomarkers related to follicle development and maturation. After searching the protein database, some proteins that are known to be involved in the development and maturation of oocyte were identified, such as apolipoprotein A-I, collagen type IV, integrin, et al. Identification of such proteins in our experiment further proved that the direct analysis of tryptic digests could be of practical value.