基因芯片技术检测鉴定临床常见致病真菌的初步研究

目的为了快速、简便、高通量地鉴定临床常见致病真菌,建立了一种采用基因芯片技术对临床常见的致病真菌鉴定的分子生物学方法。方法以5.8S rDNA与28S rDNA间的内转录间区2(internal transcribed spacer-2,ITS-2)为靶标,针对待检的临床常见致病真菌设计合成一系列寡核苷酸探针,制成寡核苷酸芯片。待检真菌DNA经通用引物扩增标记后,与芯片杂交,对杂交图谱分析归纳,得到一套种特异性的典型杂交图谱。待检的样品菌与基因芯片杂交,得到的杂交结果与典型图谱比对即可判断出样品的种类。结果以涉及8个属20个种的标准致病真菌菌株对芯片的特异性、重复性、灵敏度进行考察,结果表明,该研究建立的基因芯片技术可以有效地区分20种临床常见致病真菌,特异性良好,重复性良好(信噪比CV<10%),灵敏度为15 pg/ml真菌DNA。收集从临床分离的84株致病真菌菌株对基因芯片进行试用,结果显示基因芯片的鉴定结果与常规鉴定方法的鉴定结果一致。结论这项技术的建立可以稳定、特异性地实现临床常见致病真菌的高通量鉴定,为进一步检测研究奠定了基础。     

基因芯片技术在乙型肝炎病毒基因分型检测中的应用探讨

目的：探讨利用基因芯片技术建立乙型肝炎病毒基因分型诊断方法的可能性。方法：查阅国际上主要的乙型肝炎病毒(HBV)基因分型诊断标准和现行技术，通过将基因芯片技术与目前用于HBV基因分型的技术方法进行对比，探讨利用基因芯片技术建立乙型肝炎病毒基因分型诊断方法的可能性。结果：HBV的基因分型，既可通过全基因序列比对，也可通过片段基因序列比对，片段基因序列比对是实际工作中HBV基因分型的主要方法，现有报道的技术主要为型特异引物(SSP)PCR扩增法和PCR扩增型特异探针(SSO)杂交法。基因芯片技术具有高敏感、高通量和能够平行检测DNA类型等特点，技术类型属于PCR扩增型特异探针(SSO)杂交法。尚未见有应用基因芯片技术进行HBV基因分型检测的报道。结论：借鉴现有的PCR扩增型特异探针（SSO)杂交法，如微板核酸杂交一ELISA显色技术，理论上利用基因芯片技术建立乙型肝炎病毒基因分型诊断的方法是完全可能的，并且具有高效、快速和廉价等特点。     

通用型病毒检测芯片在三种人类致病病毒属检测中的应用

目的 研究制备对人类有致病作用的38个属的病毒寡核苷酸（oligo）基因芯片对三种人类致病病毒的检测能力。方法 应用生物信息学软件设计70-mer oligo探针，固定于玻片载体制备成基因芯片。以痘苗病毒天坛株、甲肝病毒、乙肝病毒作为检测样本，提取病毒核酸，经随机引物扩增、荧光标记，用于芯片杂交，清洗和干燥后对芯片进行扫描和数据分析。结果 芯片上oligo探针能与相应病毒的PCR扩增样品杂交，呈现阳性荧光信号。结论 建立的病毒寡核苷酸基因芯片能够检出和区分三种检测病毒，为进行未知病毒的基因芯片筛查方法的建立奠定了基础。     

6种常见呼吸道感染细菌16s rRNA基因的克隆与鉴定

目的：克隆并鉴定6种常见呼吸道感染细菌16srRNA基因，为制备基因芯片探针做准备。方法：设计、合成6种细菌16srRNA基因扩增引物；PCR扩增16srRNA基因目的片段；克隆16srRNA基因片段；最后对重组质粒进行鉴定。结果：（1）6种细菌16srRNA基因片段的PCR扩增结果：大肠埃希菌、金黄色葡萄球菌、肺炎链球菌、肺炎克雷伯杆菌、流感嗜血杆菌在1300bp处出现特异的目的片段；铜绿假单胞菌在1100bp出现特异的目的片段，与预计的片段大小吻合。（2）PCR产物分别与pMD18-T载体连接并转化JM109受体菌之后在Amp^r培养基表面生长，其中蓝色菌落为阴性克隆，白色菌落是阳性克隆。（3）各克隆质粒PCR鉴定及双酶切鉴定，结果均可见特异目的带。（4）克隆质粒的序列分析示：6种细菌克隆质粒部分16srRNA基因序列与GenBank中发表序列同源性为99．8％以上，说明细菌16srRNA基因已克隆成功。结论：成功克隆了大肠埃希菌、金黄色葡萄球菌、肺炎链球菌、肺炎克雷伯杆菌、流感嗜血杆菌及铜绿假单胞菌16srRNA基因片段，为制备基因芯片探针奠定了基础。     

基因芯片法特异性检测丙型肝炎病毒的基因分型

目的：采用基因芯片特异性检测血清中丙型肝炎病毒（HCV）并进行基因分型。方法：设计HCV基因型特异探针，将其固定在玻璃片上制成微阵列芯片。阳性组血清60份，阴性组血清15份，乙型肝炎血清5份（抗HCV阴性）。经核酸提取，多聚酶链式反应（PCR）扩增，与芯片上的探针杂交，最后分析结果并与测序分型结果比较。结果：阳性组血清全部检测到HCV-RNA，均有基因芯片分型结果。基因芯片分型结果与测序分型结果一致者56例。阴性组血清HCV-RNA全部阴性。乙型肝炎血清全部阴性。结论：基因芯片可准确对HCV感染血清做定性检测并同时检测HCV基因型，简便快捷，特异性好，并且不需荧光标记和昂贵的荧光扫描仪器，与乙型肝炎血清无交叉反应。可替代基因测序分型，适于临床大量样品的检测。     

贝氏柯克斯体毒力相关基因芯片对其他胞内寄生菌基因组DNA检测结果分析

目的用建立的贝氏柯克斯体毒力相关基因芯片研究贝氏柯克斯体与其致病性相似的专性胞内寄生菌（立克次体、埃立克体、新立克次体）,以及与其密切相关的兼性胞内寄生菌（巴通体和肺炎军团菌）等病原菌的毒力基因的差异。方法贝氏柯克斯体国际标准株-九里株作为本研究参考株,从全基因序列中选取166个毒力及毒力相关基因进行PCR扩增,纯化后的产物制备基因芯片。采用双色荧光标记,待测DNA（即用于分析的各菌株基因组DNA）用Cy5标记,贝氏柯克斯体九里株参照基因组DNA用Cy3标记。芯片杂交后,采用软件GenePixPro4.1,辅助以软件Excel,进行图片处理和数据分析。结果芯片的166个基因片段中有165个存在于所有贝氏柯克斯体属的6个分离株中,另一锚定蛋白（AnkI）基因（CBU1213）仅存在于九里株及Grita株;立克次体属的5株菌株均与CBU1631和CBU1125杂交;埃立克体属的3种菌株与新立克次体属的3种菌株均含有基因CBU1125;东方属的1株菌株具有基因CBU1449,而巴通体属的菌株,军团菌和大肠杆菌与基因芯片均无杂交。结论贝氏柯克斯体种内基因组具有高度保守性,它的专性细胞内寄生菌的毒力相关基因与胞外寄生菌的毒力相关基因有非常显著差异。     

HLA-DR位点的基因芯片分型与临床应用

目的：建立一种新型的HLA－DR位点的基因分型方法，为HLA－DR的基因分型提供一个较新的思路。方法：利用基因芯片技术HLA不同基因亚型的独特序列设计探讨，制成分型芯片；待检测样品经PCR反应标记上荧光之后，与芯片进行杂交，根据杂交产生的荧光信号值分析确定样品DR位点的基因亚型，将这一方法应用于70份标准DNA和200份医院移植供受者的HLA－DR基因分型并将部分样品进行基因测序。结果：检测结果表明HLA－DR基因分型芯片可准确分辨出DR位点等位基因30大类，耗时3h 。结论：基因芯片用HLA－DR的基因分型，分辨率高，特异性强，重复性好，操作简便，对比常规的PCR－SSP和PCR－SSO方法，HLA－DR基因芯片方法更为直观，并具有集成化优势。可以在一张芯片上同时检测HLAⅠ类的A、B位点，并实现一张芯片多人份，适合于临床应用和骨髓库，脐血库的建立。     

HLA-A寡核苷酸芯片检测条什的初步探索

寡核苷酸芯片（Oligochip）是近年来发展在熟的一种高通量基因检测技术。寡核苷酸基因芯片的一个主要用途是基因突变的快速和高通量检测。本研究以HLA小型阵列为平台，探讨寡核苷核芯片检测条件（样品制备方法和单链扩增条件）的简化和优化。     

基于DNA芯片的细菌基因表达谱技术的建立与评价

目的建立和优化基于全基因组DNA芯片的细菌基因表达谱技术，并用实时定量RT-PCR对此技术平台进行评价。方法提取并纯化鼠疫耶尔森菌总RNA，以六核苷酸随机引物（N6）和／或基因组特异引物（GDP）逆转录合成cDNA，用CY染料直接或间接标记后，与包括鼠疫耶尔森菌4005个ORF的全基因组芯片杂交，通过芯片扫描仪获得表达谱分析结果，归一化后进行分析。结果用实时定量PCR技术验证。结果采用N6＋GDP混合引物以间接法标记获得了较为理想的结果，表达谱技术与实时定量PER技术所得结果高度相关，证明了此技术的可靠性和实验设计与数据分析的合理性。结论合理的设计和归一化的分析方法是基于DNA芯片的细菌基因表达谱分析成功的关键，该技术的建立为细菌功能基因组研究奠定了基础。     

基因芯片技术在关节软骨研究中的应用进展

基因芯片通常指DNA芯片,其基本原理是将指大量寡核苷酸分子嗣定于支持物上,然后与标记的样品进行杂交,通过芯片扫描检测杂交信号的强弱进而判断样品中靶分子的数量。基因芯片能对微量样品中的核苷酸序列进行检测和分析,其高通量、快速、并行化采集生物信息的特点均优于其他传统基因检测技术,故已广泛应用于医学各领域研究。将近年来基因芯片技术在关节软骨生物学特性、形成及发育、损伤与修复、退变及再生等领域的应用进行综述：     

Parallel Detection of Bacterial Pathogens in Cerebrospinal Fluid with 16S rRNA Probe Microarray

The most commonly used method for detection ofpathogenic bacteria in cerebrospinal fluid (CSF) speci mens of clinical laboratories is isolation and identificationof the causative agents by cultural method, biochemicaland serological t…     

Use of a three-dimensional microarray system for detection of levofloxacin resistance and the mec A gene in Staphylococcus aureus

We evaluated a novel three-dimensional microarray (Pam Chip microarray) system to detect the presence of levofloxacin-related resistance mutations and the mec A gene. The results were compared to those obtained for 27 Staphylococcus aureus isolates by conventional DNA sequencing or PCR methods. Hybridization and fluorescence detection were performed using an FD 10 system designed for Pam Chip microarray under conditions optimized for each target/probe on the array. In dilution series analysis using multiplex PCR samples, the sensitivity of the microarray was about 10 times greater than that of conventional PCR methods. A high level of data reproducibility was also confirmed in those analyses. Various point mutations in quinolone resistance-determining regions detected by our system corresponded perfectly to the results obtained by conventional DNA sequencing. The results of the mec A gene detection using our system also corresponded to the PCR method; that is, signal/band was detected in all isolates of methicillin-resistant S. aureus, and no signal/band was detected in any isolate of methicillin-susceptible S. aureus. In conclusion, our novel three-dimensional microarray system provided rapid, specific, easy, and reproducible results for the simultaneous detection of levofloxacin resistance and the mec A gene in S. aureus.     

APC基因甲基化定量检测芯片的建立

目的 建立抑癌基因APC（adenomatous polyposis coli，APC）启动子1A的甲基化定量芯片检测方法。方法选取一段420bp的APC基因启动子1A CpG密集序列作为靶序列，针对M0、M1、M2、M3、M4 5个CpG靶位点，设计一套检测甲基化与非甲基化的探针。采用脐带血DNA克隆体作为阴性、阳性质控品。结果甲基化阳性、阴性质控的芯片结果与测序吻合。每组探针中荧光强度由强至弱依次为，阳性质控（甲基化）：探针1〉2、3〉4；阴性质控（非甲基化）：探针3〉4、1〉2。5个位点的5条荧光强度标准曲线，尺。范围是0．93～0．99。M0、M1、M2、M3、M4 5个位点甲基化杂合型的检测范围分别为50．0％±3．6％、50．0％±6．9％、50．0％±3．5％、50．0％±8．5％、50．0％±7．3％。结论建立了APC基因启动子5个COG位点的甲基化定量检测芯片。     

Simultaneous analysis of foodborne pathogenic bacteria by an oligonucleotide microarray assay

A rapid and accurate method for simultaneous identification of foodborne infectious pathogens was developed based on oligonucleotide microarray technology. The proposed identification method is based on PCR amplification of the target region of the groEL genes with degenerate primers, followed by the PCR products hybridization with oligonucleotide probes specific for species. The groEL gene amplification products of seventeen species of pathogenic bacteria were hybridized to the oligonucleotide array. Hybridization results were analyzed with digoxigenin-linked enzyme reaction. Results indicated that fifteen species of pathogenic bacteria showed high sensitivity and specificity for the oligonucleotide array, while two other species gave cross-reaction with the E. coli. Our results suggested that microarray analysis of foodborne infectious pathogens might be very useful for simultaneous identification of bacterial pathogens. The oligonucleotide array can also be applied to samples collected in clinical settings of foodborne infections. The superiority of oligonucleotide array over other tests lies on its rapidity, accuracy and efficiency in the diagnosis, treatment and control of foodborne infections.     

An integrated algorithm for gene selection and classification applied to microarray data of ovarian cancer

OBJECTIVE: The type of data in microarray provides unprecedented amount of data. A typical microarray data of ovarian cancer consists of the expressions of tens of thousands of genes on a genomic scale, and there is no systematic procedure to analyze this information instantaneously. To avoid higher computational complexity, it needs to select the most likely differentially expressed gene markers to explain the effects of ovarian cancer. Traditionally, gene markers are selected by ranking genes according to statistics or machine learning algorithms. In this paper, an integrated algorithm is derived for gene selection and classification in microarray data of ovarian cancer. METHODS: First, regression analysis is applied to find target genes. Genetic algorithm (GA), particle swarm optimization (PSO), support vector machine (SVM), and analysis of variance (ANOVA) are hybridized to select gene markers from target genes. Finally, the improved fuzzy model is applied to classify cancer tissues. RESULTS: The microarray data of ovarian cancer, obtained from China Medical University Hospital, is used to test the performance of the proposed algorithm. In simulation, 200 target genes are obtained after regression analysis and six gene markers are selected from the hybrid process of GA, PCO, SVM and ANOVA. Additionally, these gene markers are used to classify cancer tissues. CONCLUSIONS: The proposed algorithm can be used to analyze gene expressions and has superior performance in microarray data of ovarian cancer, and it can be performed on other studies for cancer diagnosis.     

mi R-22在压应力下软骨终板细胞中的表达及其对靶基因MMP-1的调控作用

目的 应用miRNA芯片技术筛选压应力下人软骨终板细胞中差异表达的miRNAs,研究与压应力相关的miR-22并进行生物信息学分析,探讨其对靶基因MMP-1的调控。方法 将6个月意外流产胎儿软骨终板进行软骨终板细胞的分离、培养。将第2代软骨细胞按实验要求种板培养并随机分为加压组与对照组。加压组施加周期性压力(5 MPa,6 h×5 d),使用TRIzol法提取各组RNA,miRNA芯片检测miRNA表达情况,然后用Realtime PCR(RT-PCR)对差异表达miR-22进行验证。通过靶基因预测软件预测MMP-1与miR-22基因的关系,miR-22 mimics脂质体转染Hela细胞后通过RT-PCR进一步验证。结果 芯片扫描结果显示加压组miR-22下调明显。RT-PCR检测显示压应力下人软骨终板细胞miR-22表达显著降低。miR-22 mimics转染目标细胞48 h后,miR-22的表达显著增高,同时MMP-1基因表达显著降低。结论 压应力下miR-22异常表达,miR-22可靶向负调控与软骨终板细胞退变相关的MMP-1基因。     

Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray

We coupled multiplex PCR and a DNA microarray to construct an assay suitable for the simultaneous detection of five important marine fish pathogens (Vibrio vulnificus, Listonella anguillarum, Photobacterium damselae subsp. damselae, Aeromonas salmonicida subsp. salmonicida, and Vibrio parahaemolyticus). The array was composed of nine short oligonucleotide probes (25-mer) complementary to seven chromosomal loci (cyt, rpoN, gyrB, toxR, ureC, dly, and vapA) and two plasmid-borne loci (fatA and A.sal). Nine primer sets were designed to amplify short fragments of these loci (100 to 177 bp) in a multiplex PCR. PCR products were subsequently labeled by nick translation and hybridized to the microarray. All strains of the five target species (n = 1 to 21) hybridized to at least one species-specific probe. Assay sensitivities ranged from 100% for seven probes to 83 and 67% for the two remaining probes. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria (n = 40 strains; 100% specificity). Using purified genomic DNA, we were able to detect PCR products with < 20 fg of genomic DNA per reaction (equivalent to four or five cells), and the array was at least fourfold more sensitive than agarose gel electrophoresis for detecting PCR products. In addition, our method allowed the tentative identification of virulent strains of L. anguillarum serotype O1 based on the presence of the fatA gene (67% sensitivity and 100% specificity). This assay is a sensitive and specific tool for the simultaneous detection of multiple pathogenic bacteria that cause disease in fish and humans.     

Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria

A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.