DNA微阵列技术检测HLA-DRB1基因分型

目的采用DNA微阵列分型方法,对HLA-DRB1基因进行分型研究。方法设计分型检测寡核苷酸探针,制备HLA-DRB1基因分型微阵列。设计PCR引物,以1:20引物比例不对称扩增HLA-DRB1基因的第2外显子,Cy3荧光标记扩增产物,作为杂交模板。用差异选择法杂交信号强且特异性好的探针。Cy3荧光标记PCR扩增产物与微阵列探针杂交反应,结果经荧光扫描,并用分型软件分析判断阳性探针和HLA-DRB1基因型。结果10例临床血样的DNA微阵列分型结果与PCR-SSP分型结果相符,20例未知血样的DNA微阵列分型结果与DNA测序分型结果符合率为97%。结论DNA微阵列技术具有高通量、简便、低成本的优势,采用DNA微阵列分型方法,对HLA-DRB1基因进行分型研究,表明HLA-DRB1DNA微阵列分型为一种可行的基因分型新技术。     

膜反向点杂交技术在HIV-1耐药性点突变检测中的应用

目的以HIV基因组pol区序列为靶基因设计可鉴别HIV耐药性突变的寡核甘酸探针对，应用膜反向点杂交技术检测HIV-1耐药性点突变。方法以临床提取HIV-1患者血清为模板，扩增产物与点在尼龙膜上的5对特异的寡核苷酸探针杂交。通过标记在PCR上游引物5’端的荧光素显色得到结果，并将其与测序结果进行对比。结果采用反向点杂交共检测了9例野生型和21例突变耐药型样本，检测结果与测序结果符合率为74％。5对探针对于野生型和突变型的扩增产物均有较好的区分性，敏感性高且信号强弱与相应产物在样本所占比例相关。结论应用膜反向点杂交技术检测HIV耐药性点突变敏感、简便、快速，具有较高应用价值。     

寡核苷酸芯片用于HLA-A基因分型的研究

目的 应用寡核苷酸芯片分型方法，对HLA-A基因进行分型研究。方法 采用不对称PCR方法，扩增HLA-A基因的第2，3外显子，荧光标记扩增产物，作为杂交模板。设计分型检测寡核苷酸探针，制备HLA-A基因分型检测芯片。采取差异选择法，筛选强杂交信号和高特异性的分型探针。探索了探针长度、探针位置等对杂交信号的影响。杂交结果经荧光扫描，并用分型软件分析判断阳性探针和HLA-A基因型。结果 30例临床样本芯片分型结果与PCR-SSP及DNA测序分型结果相符。结论 采用寡核苷酸芯片技术对HL-A基因分型是种好的方法，具有测速快、成本低、高通量的优点。     

乙型脑炎病毒寡核苷酸基因芯片研究

目的:制备检测乙型脑炎病毒寡核苷酸(oligo)基因芯片。方法:应用生物信息学软件设计60-meroligo探针用于制备基因芯片,乙型脑炎病毒(JEV)基因片段经限制性显示技术扩增标记,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:大部分oligo探针都能特异性与相应样品杂交,呈现阳性荧光信号,而阴性对照和空白对照则基本不能检测到荧光信号。结论:实验中建立的oligo基因芯片检测病原体方法可行,具有应用于临床诊断的前景。     

人白细胞抗原B位点基因芯片分型技术研究

目的 探讨基因芯片技术在进行北方汉族人群人白细胞抗原B位点（HLA-B）分辨度分型的价值.方法 根据中国北方汉族人群HLA-B常见基因位点及临床分型分辨度特征,设计特异性寡核苷酸中分辨度分型探针,制成HLA-B基因分型芯片.采用荧光标记引物和不对称聚合酶链反应（PCR）扩增HLA-B 2、3外显子,产物与芯片探针杂交后经荧光扫描,并用特定软件分析判断阳性探针,以确定样品基因型.结果 用中分辨度探针从30份北方汉族人标本中可分出HLA-B 7～83范围的42个B抗原等位基因,与顺序特异引物聚合酶链反应（PCR-SSP）分型方法对比,多检出3个HLA-B14、73和82新等位基因.结论 HLA-B基因芯片具有较高的精确度和特异性,可一张芯片多人份检测,适合用于临床HLA-B抗原分型.     

HCV基因型检测芯片的设计与研制初报

目的：设计与研制丙型肝炎病毒（HCV）基因型检测芯片。方法：用生物信息学方法设计HCV 5'UTR型特异性探针。按设计模式，以自动化微阵列点样仪将探针点样于APTES－PDC修饰的玻片表面。用商品PCR试剂盒掺入荧光分子对患者血清样本进行扩增，用激光共聚焦荧光扫描仪检测并分析结果。结果：根据5'UTR序列设计了各型HCV的特异性探针。芯片表面修饰符合固定探针和杂交检测的需求。含荧光分子的RT－PCR产物可成功地与固定于芯片上的探针阵列杂交进行HCV基因型的检测。21例丙型肝炎患者中，19例为1b型，1例为2a型，1例为混和型。结论：所设计研制的基因芯片可望成为检测HCV基因型的操作简便，结果准确，价格低，耗时短的新型实验室检测方法。     

生物素链霉亲和素系统基因芯片检测ApoE基因多态性

目的建立一种准确、简便、低成本的临床实用型Apo E基因多态性芯片检测技术。方法通过基因文库获取Apo E基因全序列;以Primer Primer 5.0设计探针和引物,oligo6验证。摸索PCR扩增条件,产物测序确认;质粒构建并测序确认。探针点样在含链亲蛋白的基因芯片(专利技术)上,待检样品以生物素d UTP标记3'端进行扩增,然后与基因芯片杂交洗脱,阳性结果为纳米金紫红色的圆点状,肉眼即可判断实验结果。以本法检测160例临床样本,并进行测序验证。结果基因芯片检测完毕后通过肉眼识别紫色点状(生物素-链霉亲和素系统biotin-streptavidin systems,BSAS)分布位置,由此可准确分辨Apo E基因型。临床样本实测结果与测序结果具高度一致性(kappa=0.971,P0.001)。结论在具备标准基因实验室的条件下即可应用此技术进行Apo E基因多态性分析,可临床常规应用。     

细菌性肺炎快速诊断基因芯片的初步构建

目的：根据细菌16 S rRNA基因特点设计常见病原菌的特异性探针,采用酶显色技术构建基因芯片,探讨其临床应用的可能性。方法选取肺炎链球菌、流感嗜血杆菌及铜绿假单胞菌等8种细菌性肺炎常见的病原菌的标准菌株作为研究对象,并选择12份患者的痰液标本进行检测。在16 S rRNA基因保守区设计 PCR反应的通用引物及革兰阳性菌、革兰阴性菌的通用探针,利用可变区的差异设计合成特异性探针,构建基因芯片。利用细菌16S rRNA基因设计的PCR通用引物进行扩增,所有8种细菌均获得350 bp的扩增产物。以地高辛标记特异性探针,构建完成可用于8种常见病原菌检测的基因芯片,结果8种标准菌株基因芯片检测均取得了预期效果,对12份痰标本中常规培养阳性7份,其对应芯片检测结果均成阳性,5份常规培养阴性的标本中,芯片结果提示阳性的有3份,其中1份为嗜肺军团菌,2份为使用抗生素后的患者标本。结论设计合成的 PCR通用引物对扩增细菌的16 S rRNA基因具有较高的特异性及灵敏度。构建的基因芯片可用于常见细菌性肺炎病原菌的检测鉴定,且对抗生素使用后的临床标本及苛养菌有一定的诊断价值。本研究所获得基因芯片对于细菌性肺炎的检测具有简单、快速、特异性及敏感性高的特点。     

基因芯片技术检测临床常见致病性念珠菌和新型隐球菌及ERG11基因点突变的实验分析

目的建立基因芯片技术检测临床常见致病性念珠菌和新型隐球菌及氟康唑耐药白念珠菌相关ERG11基因点突变的试验方法。方法针对临床常见的5种致病性念珠菌和新型隐球菌5.8S r DNA与28S r DNA间的内转录间区2(ITS-2)基因设计、合成一系列寡核苷酸探针,制备寡核苷酸芯片,以鉴定5种致病性念珠菌和新型隐球菌;设计、合成能够特异性扩增ERG11基因点突变的引物,并采用不对称荧光聚合酶链反应(PCR)扩增ERG11基因,将PCR产物与芯片进行杂交。结果采用特异性引物和不对称荧光PCR从12株临床分离耐药株中成功扩增出ERG11基因4个突变点;采用基因芯片杂交技术成功鉴定5种临床常见致病性念珠菌和新型隐球菌。结论制备的寡核苷酸基因芯片,可以用于鉴定临床常见的致病性念珠菌和新型隐球菌。     

基因芯片检测输血传播病毒方法的实验研究

目的建立基因芯片快速检测经输血传播病毒核酸的方法,进而探讨该方法用于检测临床标本的可行性。方法通过PCR获得TTV病毒ORF1基因的DNA片段,克隆,从重组质粒扩增DNA片段,并点到玻璃载体上,制成芯片。与TTV病毒、甲型肝炎病毒、乙型肝炎病毒及戊型肝炎等病毒的PCR产物进行杂交,以检测探针的特异性。结果该基因芯片探针仅与TTV毒株的PCR产物杂交呈阳性,与对照病毒的PCR产物杂交呈阴性。敏感性试验显示,用该方法检测了27份疑似TTV临床病料,22份阳性;而用PCR法扩增TTV ORF1基因确诊为阳性的只有19份。结论利用基因芯片检测TTV的PCR产物,特异性和敏感性强,可作为TTV临床标本检测方法。     

Towards specific diagnosis of plant-parasitic nematodes using DNA oligonucleotide microarray technology: a case study with the quarantine species Meloidogyne chitwoodi

In order to investigate the feasibility of a microarray-based method for diagnostics of plant-parasitic nematodes, we have developed a DNA oligonucleotide microarray to detect the nematode species Meloidogyne chitwoodi, which is listed as a quarantine organism in Europe. Oligonucleotide capture probes were designed from nematode SCAR and satellite DNA sequences and spotted onto epoxy-coated glass slides. PCR products were generated using specific primers, labeled with Cyanine 3 or Cyanine 5 fluorescent dyes, and hybridized overnight to the microarray. This methodology allowed the specific detection of M. chitwoodi DNA in pure and mixed samples (i.e. when M. chitwoodi DNA was mixed with DNA from a congeneric nematode species). Simultaneous hybridization of the microarray with two amplified targets labeled with different dyes proved to be efficient, without any competition between the targets. These results illustrate a significant step forward in the development of the DNA chip technology for nematode detection, and constitute to our knowledge the first report of production and use of oligonucleotide microarrays for the detection of plant-parasitic nematodes, using the quarantine species M. chitwoodi as a test organism.     

建立检测细胞色素P450酶与紫杉醇代谢相关突变位点的基因芯片分型方法

目的 建立针对与临床抗癌药物紫杉醇代谢相关的细胞色素P450(CYP450)酶基因多态性位点的快速、准确、高通量的基因芯片基因分型方法.方法 选取CYP450酶2C8*3、3A4* 18、3A5*3C等3个与紫杉醇代谢相关突变位点,根据基因库中报道的序列,设计每个基因多态性位点的野生型和突变型探针,在突变位点2侧设计PCR扩增引物,PCR片段长度应＜200 bp,并构建标准质粒.探针在3'端氨基修饰,下游引物标记荧光素Cy3,将探针按一定顺序点样于经醛基化处理的玻片制备成基因芯片.人体血液DNA样本分别通过3对引物扩增后与基因芯片上的探针杂交,通过扫描图像和配套软件对结果进行分析和判断.同时,对50份血液样本进行检测.结果 通过标准质粒杂交结果显示,每个位点对应的1对探针均能将野生型和突变型质粒进行准确地区分,无非特异性杂交信号出现;检测50份血液样本,CYP2C8 * 3位点的突变率为2%,CYP3A4 * 18位点均为野生型,CYP3A5 * 3C位点的突变率为62%.同时,通过测序法进行验证,基因芯片方法与测序方法的结果完全一致.结论 建立的同时检测抗癌药物紫杉醇代谢相关的CYP450酶基因多态性位点CYP2C8*3、CYP3A4 * 18、CYP3A5 * 3C的基因芯片分型方法快速、准确,结果可靠,重复性好,可用于指导紫杉醇患者的个体化用药,并为分析个体患者对于紫杉醇药物体内代谢提供了一个高通量技术平台.     

Using PCR amplification to increase the confidence level of Salmonella typhimurium DNA microarray chip hybridization

In order to design and validate a method to identify virulence genes of Salmonella typhimurium using DNA microarray, a protocol was developed to label the isolated bacterial DNA directly and to use PCR amplification of limited numbers of genes to validate the hybridization signals. Therefore, a DNA microarray chip of 71 virulence genes of S. typhimurium was developed and evaluated using 10 isolates. Each gene was represented by 65bp oligonucleotide probes (oligoprobes) and immobilized on the surface of chemically modified slides. Whole DNA genomes were digested with Hinf1 and Sau3AI, labeled with a fluorescent tag of Cy3 and then hybridized. The presence of virulence genes in 10 strains of S. typhimurium was established by measuring a fluorescent signal above the background noise of the chip. PCR amplification of 10 genes (orgA, ORF319, ttrB, rmbA, misL, spi4F, spi4H, spi4N, rRNA, and purR) of S. typhimurium was used as a standard to verify the confidence level of the DNA microarray chip. In conclusion, using PCR amplification to increase the confidence level of the microarray hybridization data was successful.     

Genotyping of Clostridium perfringens toxins using multiple oligonucleotide microarray hybridization

A microarray-based method for characterization of six Clostridium perfringens toxin genes: iA (iota toxin), cpa (alpha toxin), cpe (enterotoxin E), etxD (epsilon toxin), cpb1 (beta toxin 1),and cpb2 (beta toxin 2) was developed and evaluated using 17 C. perfringens isolates. Three individual oligonucleotide probes (oligoprobes), complementary to the unique sequences of each toxin gene, were designed and immobilized on a surface of aldehyde-coated glass slides. Multiplex PCR was used to simultaneously amplify DNA target regions of all six genes. Single-stranded DNA (ssDNA) samples for microarray analysis were prepared by following a primer extension of amplicons in the presence of one primer. Fluorescent moieties (Cy3) were incorporated into the ssDNA by chemical modification of guanine bases. The presence of toxin genes in C. perfringens was established by hybridization of the fluorescently labeled ssDNA representing different samples to the microarray gene-specific oligoprobes. Results of the study showed sensitivity and specificity of genotyping C. perfringens using multiple microarray-based assays.     

检测拉米夫定耐药位点基因芯片的研制及其应用初探

目的：研发检测拉米夫定耐药的基因芯片进行临床拉米夫定致乙型肝炎病毒（HBV）耐药相关基因突变的监测。方法：（1）将HBV YMDD区4个突变位点为靶的16条寡核苷酸探针，用GMS 417芯片点样仪固定在经特殊处理的玻片上。待检HBV突变相关的核酸经过聚合酶链反应（PCR）扩增，及用Cy5标记的三磷酸脱氧胞苷进行荧光标记，再通过与基因睛杂交，严谨洗涤，将非突变的标记片段洗脱后，将芯片在Gene TAC LS IV扫描仪下进行扫描，计算机解读。（2）应用PCR定点突变技术构建Leu515Met,Met539Ile,Met539Val和V542I 4位点和4位点的单一突变质粒，并将芯片检测结果与测序结果对照，以鉴定其特异性。同时，用血清标本和质粒的重复检测，测定其重要性，并对50份拉米夫定治疗血清和慢性HBV感染患者血清进行检测。结果：我们研制的芯片能同时特异性地检测耐药相关单一或多位点突变。其检测与测序的符合率98％。重复率达96％－100％。结论：本研究制作的4位点基因芯片，既可检测乙型肝炎拉米夫定耐药相关单一碱基突变，亦可一次有效检出4个位点的突变，且具有快速、高特异性和可重复性，检测结果可靠的优点。     

幽门螺杆菌克拉霉素耐药基因芯片的制备和应用

目的 建立一种寡核苷酸微阵列检测幽门螺杆菌23S rRNA基因A2142G、A2143G及C2182T点突变的方法.方法 根据23S rRNA基因A2142G、A2143G及C2182T突变位点设计相应探针,样本经不对称PCR扩增后,其产物与芯片杂交.非荧光标记引物扩增PCR产物克隆至T载体,测序验证芯片结果,并结合临床最低抑菌浓度实验判断该方法的正确性.结果 寡核苷酸微阵列技术与测序检测幽门螺杆菌23S rRNA基因多态性结果完全一致.经培养及鉴定幽门螺杆菌阳性的54份标本,杂交结果显示A2142位点均为野生型(54/54);A2143G突变率为11.11%(6/54),尚未发现A2143C和A2143T的突变;C2182T突变率为12.96%(7/54),尚未发现C2182A和C2182G的突变,其余均为野生型,上述结果与菌株体外试验MIC结果完全一致.结论 建立一种寡核苷酸微阵列技术检测幽门螺杆菌克拉霉素耐药的23S rRNA基因多态性的方法,可以高通量并直接检测胃黏膜而不需进行细菌培养,推动个体化治疗方案的实施.     

Molecular identification of Yersinia enterocolitica isolated from pasteurized whole milk using DNA microarray chip hybridization

A DNA microarray chip of four virulence genes and 16S ribosomal DNA gene conserved region among all Gram negative species, including Yersinia, as a positive control was developed and evaluated using 22 Yersinia enterocolitica isolates. Eight different oligonucleotide probes (oligoprobes) with an average size of 22 bp, complementary to the unique sequences of each gene, were designed and immobilized on the surface of chemically modified slides. Multiplex PCR was used to simultaneously amplify DNA target regions of all five genes, and single stranded DNA (ssDNA) samples for microarray analysis were prepared by using a primer extension of amplicons in the presence of one primer of all genes. The presence of genes in Y. enterocolitica was established by hybridization of the fluorescently labeled ssDNA representing different samples of the microarray gene-specific oligoprobes and confirmed by PCR. Results of the study showed specificity of genotyping Y. enterocolitica using multiple microarray-based assays. Final validation of the chip's ability to identify Y. enterocolitica genes from adulterated pasteurized whole milk was confirmed and successful. The limit of chip detection of virulence genes in pasteurized whole milk was found to be 1000 CFU per hybridization.     

纳米金比色芯片同步目视化检测HBV和HCV

目的 建立一种简单、快速的比色芯片可同时目视化检测HBVDNA和HCVRNA。方法 利用多重PCR方法扩增HBsAg阳性合并抗HCV阳性的患者血清中提取的HBVDNA和HCVcDNA。制备Au纳米颗粒基因探针，检测探针与固定在尼龙膜上的捕捉探针构成双探针，用斑点杂交法检测HBVDNA、HCVcDNA的多重PCR产物，加入银离子（Ag^＋）-对苯二酚液染色观察结果。结果 多重PCR可同时扩增HBVDNA和HCVcDNA，出现预期的431bp和323bp特异性条带。纳米金比色芯片可以同步目视化检出乙型、丙型肝炎合并感染患者血清中HBVDNA和HCVRNA的PCR产物。结论 目视化HBV、HCV纳米金比色芯片诊断方法操作简单、特异性好、成本低廉、结果 判定指标客观，此类基因芯片在病毒基因检测领域将会有广泛用途。     

Sequence-specific identification of 18 pathogenic microorganisms using microarray technology

We have developed a Multi-Pathogen Identification (MPID) microarray for high confidence identification of eighteen pathogenic prokaryotes, eukaryotes and viruses. Analysis of amplified products from pathogen genomic DNA using microarray hybridization allows for highly specific and sensitive detection, and allows the discrimination between true amplification products and false positive amplification products that might be derived from primers annealing to non-target sequences. Species-specific primer sets were used to amplify multiple diagnostic regions unique to each individual pathogen. Amplified products were washed over the surface of the microarray, and labelled with phycoerythrin-streptavidin for fluorescence detection. A series of overlapping 20-mer oligonucleotide probes hybridize to the entire diagnostic region, while parallel hybridizations on the same surface allow simultaneous screening for all organisms. Comparison to probes that differ by a single mismatch at the central position reduced the contribution of non-specific hybridization. Samples containing individual pathogens were analyzed in separate experiments and the corresponding species-specific diagnostic regions were identified by fluorescence among their highly redundant probe sets. On average, 91% of the 53 660 pathogen probes on the MPID microarray performed as predicted. The limit of detection was found to be as little as 10 fg of B. anthracis DNA in samples that were amplified with six diagnostic primer-pairs. In contrast, PCR products were not observed at this concentration when identical samples were prepared and visualized by agarose gel electrophoresis.     

DNA microarray for the detection of therapeutically relevant antibiotic resistance determinants in clinical isolates of Staphylococcus aureus

An oligonucleotide microarray was constructed for the rapid and sensitive molecular detection of antibiotic resistance determinants in Staphylococcus aureus. The array is equipped with oligonucleotide capture probes for the detection of 10 clinically and therapeutically relevant antibiotic resistance genes and -mutations (mecA, aacA-aphD, tetK, tetM, vat(A), vat(B), vat(C), erm(A), erm(C), grlA-mutation) as well as several control probes. A microarray concept was established including multiplexed PCR amplification, DNA labeling, hybridization and data processing. This concept was applied to clinical Staphylococcus aureus isolates and results were concordant with those from standard genotypic and phenotypic resistance testing. Our microarray concept offers rapid and accurate identification of antibiotic resistance profiles. It is easily expandable and thus can be adapted to changing clinical and epidemiological requirements in clinical diagnosis as well as in epidemiological studies.