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研制和优化寡核苷酸芯片以初步实现对多种常见HPV(Human papillomavirus)病毒的分型检测.应用生物学软件对四型常见HPV病毒(6、11、16、18型)的全基因组序列进行分析,设计具有型特异性、熔解温度(Tm)相近的~60 mer寡核苷酸探针,对玻片片基进行优化处理后,点样制备成寡核苷酸基因芯片.将含HPV全长基因序列的质粒作为阳性标准品,利用梯度限制性荧光标记技术对其进行荧光标记,标记好的样品与芯片杂交.结果显示HPV样品与相应的型特异性探针杂交有明显的荧光信号,而与阴性对照探针和空白对照探针没有杂交信号.通过对芯片片基处理和样品荧光标记方法的优化,可以提高芯片检测的杂交特异性和荧光信号强度. 相似文献
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利用基因芯片技术筛选HIV-1F亚型基因限制性显示探针 总被引:2,自引:0,他引:2
为筛选限制性显示技术制备的HIV 1F亚型基因探针 ,应用基因芯片打印仪将其有序地打印在玻片上制备基因芯片 .在随机引物延伸的过程中进行HIV样品的荧光标记 ,然后与芯片进行杂交 .杂交后清洗玻片并干燥 ,对芯片进行扫描 ,分析各探针的杂交信号 .从中筛选了 14个基因片段作为芯片下一步研究的探针 .实验证明 ,限制性显示技术是一种制备基因芯片探针的实用方法 相似文献
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目的:利用环介导等温扩增技术(LAMP)对人乳头瘤病毒(HPV)进行快速分型检测。方法:以HPV59、HPV66和HPV68等3种病毒亚型为实验对象,选择合适的特异性核酸序列作为检测的靶序列并构建质粒,针对每个待检靶序列设计若干组扩增引物,并利用构建的质粒筛选出特异性强、灵敏度高的引物组合;将筛选成功的引物加至微流控芯片上,实现人宫颈脱落上皮细胞样本内HPV的快速核酸分型检测。结果:针对HPV59、HPV66和HPV68这3种病毒亚型,设计筛选出具有一定灵敏度的特异性引物,结合微流控芯片,无需对样本进行复杂的核酸提取纯化操作,实现了对临床样本的直接快速检测,整个检测过程在1.5 h内即可完成。结论:LAMP技术可用于临床样本中HPV的分型检测,与微流控芯片结合后操作简单、检测时间短,在临床上有较好的应用前景。 相似文献
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目的:制备乙型脑炎病毒(JEV)可视化分型基因芯片。方法:根据JEV的基因组序列,应用生物学软件设计JEV分型引物及探针,制备其可视化分型基因芯片;用生物素标记的引物PCR扩增目的片段,并与固定于玻片上的探针杂交,加入链霉亲和素标记的纳米金,银增强实现可视化;进行特异性、灵敏性及重复性试验。结果:探针特异地与相应的标记目的基因片段杂交,并在芯片上呈现较强的阳性杂交信号;2号探针能特异性检出JEV,3、4号探针可分别对Ⅰ型和Ⅲ型JEV进行分型;芯片对JEV质粒检测的灵敏度达105拷贝/mL;以蓝耳病病毒等5种病毒为对照,芯片只对JEV响应,具有特异性;制备的基因芯片具有批间、批内重复性。结论:制备的基因芯片具有高特异性、灵敏性及重复性,可以快速、准确、高通量地对JEV进行可视化分型检测。 相似文献
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生物芯片技术是上世纪90年代发展起来的新型生物技术,它具有高通量、高度集成化、微型化、高敏感、高度平行性等特点。近年来,基因芯片技术已逐渐被应用于病毒的基因分型中。病毒的基因分型芯片是直接把病毒分型探针固定在基片上从而制成的基因芯片,本文主要对病毒的基因分型芯片技术及其应用进展进行综述。 相似文献
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肺癌相关基因芯片的制作 总被引:8,自引:2,他引:6
探讨肺癌相关基因芯片的制作过程及其在基因表达谱方面的应用,同时对一些传统的技术环节进行了改进。首先有目的搜集了60个肺癌相关基因,用修饰后的引物将其分别克隆,经纯化、变性后,用Cartesian PinSys5500 cDNA Microarray点样仪以微阵列的形式将其点布于醛基化的玻璃片上;然后将人支气管上皮恶性转化细胞模型BEP2D细胞的原代、20代、35代的总RNA逆转录后再经LD PCR标记成荧光探讨与基因芯片进行杂交。ScanArray 3000荧光扫描仪扫描后显示,图象背景均匀、信号清晰,证实该芯片制作成功,并能取得良好的杂交效果。 相似文献
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甲型流感病毒流行毒株检测和分型基因芯片的研制 总被引:1,自引:0,他引:1
【目的】研制一种可同时对甲型流感病毒H1N1、H1N2、H3N2、H5N1和H9N2等5种流行亚型进行检测和分型的基因芯片。【方法】根据National Center for Biotechnology Information中Influenza Virus Resource数据库,针对H1N1、H1N2、H3N2、H5N1和H9N2等5种亚型甲型流感病毒的HA和NA基因设计46条特异性寡核苷酸探针和1条质控探针,点制成基因芯片。利用通用引物扩增流感病毒HA和NA基因,使用Klenow酶对扩增产物进行荧光标记和片段化,将标记后产物和芯片杂交,清洗、扫描后根据荧光信号判定检测结果。用18株不同种属来源的甲型流感病毒分离毒株和186份咽拭子对芯片特异性、敏感性和临床应用进行初步评价。【结果】所有18株分离毒株均能被芯片准确检测并分型,芯片检测灵敏度能达约1×104个病毒基因拷贝。同时8份咽拭子检测结果为H1N1阳性,4份咽拭子为H3N2阳性。【结论】研究表明该芯片具有较高的特异性和灵敏度,可为甲型流感病毒的监测提供一种有效的方法。 相似文献
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采用PCR扩增、pGEM T载体克隆和核苷酸序列分析的方法对一例武汉地区及两例五峰县高发区宫颈癌患者体内HPV16型的E7基因编码区进行序列分析并与野生型 (德国标准株 )及已发表的HPV16湖北株 (HPVHB)进行了比较。结果发现武汉地区HPV16型E7基因仅第 5 4位出现一个同义突变 ,而高发区HPV16型E7基因存在差异 ,第 77位氨基酸由精氨酸 (Arg)变为半胱氨酸 (Cys) ,第 96位由谷氨酰氨酸 (Gln)变为精氨酸 (Arg) ,E7蛋白的二级结构及亲、疏水性也相应改变 ,与野生型有较大差异 相似文献
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用基因表达谱芯片研究人正常肝和肝细胞癌中差异表达的基因 总被引:35,自引:0,他引:35
以基因表达谱芯片对人正常肝及肝癌组织基因表达的差异性进行了研究比较。奖4096条人cDNA用点样仪点在特制玻片上制备成表达谱芯片;利用肝和肝癌组织的mRNA通过逆转录方法,将Cy3和Cy52种荧光分别标记到两种组织的cDNA上,制备成cDNA探针,并与表达谱芯片进行杂交及扫描,重复4次实验,通过计算机数据处理判定基因是否在上述2种组织中有表达差异,筛选出差异表达的基因共903条。基因芯片技术可同时 相似文献
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We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on. 相似文献
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Normalizing DNA microarray data 总被引:1,自引:0,他引:1
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Oligonucleotide Microarray with RD-PCR Labeling Technique for Detection and Typing of Human Papillomavirus 总被引:4,自引:0,他引:4
Currently, screening for high-risk human papillomavirus (HPV) infection remains an important health concern throughout the
world, because of the close association between certain types of HPV and cervical cancer. In this study, we explore the possibility
of using ∼70mer oligonucleotide microarray for detection and genotyping of HPV. The ∼70mer type-specific oligonucleotide probes
of four different types HPV were designed by using biological software Arraydesigner 2.0, which analyzed the whole genome
sequences of HPV and selected optimal probes. These probes were synthesized and printed onto the surface of glass slides in
order to prepare a low-density microarray. HPV samples were labeled with fluorescence dyes Cy3 using a method of restriction
display polymerase chain reaction (RD-PCR). HPV plasmid DNA was restricted with Sau3A I to produce multiple fragments that
were ligated to adaptors subsequently and used as PCR template. PCR labeling was performed with the fluorescently labeled
universal primer (Cy3-UP) whose sequence is designed according to the adaptor of the RD-PCR approaches. The labeled samples
were hybridized with the oligonucleotide microarray. The scanning results showed that HPV DNA hybridized specifically with
multiple spots correspondingly to show positive signals, whereas no signals were detected of all the negative and blank controls.
These results demonstrated that ∼70mer oligonucleotide microarray can be applied to HPV detection and genotyping. The application
of RD-PCR in the sample labeling can increase significantly the sensitivity of the assay and will be especially useful for
the discriminate diagnosis of multiple pathogens. 相似文献
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Carissa M. Soto Kate M. Blaney Mubasher Dar Manzer Khan Baochuan Lin Anthony P. Malanoski Cherise Tidd Mayrim V. Rios Darlah M. Lopez Banahalli R. Ratna 《Biosensors & bioelectronics》2009,25(1):48-54
Previous studies have shown that a functionalized viral nanoparticle can be used as a fluorescent signal-generating element and enhance detection sensitivity for immunoassays and low density microarrays. In this study, we further tested this ability in commercial DNA microarrays, including Affymetrix high density resequencing microarray. Optimum conditions for NeutrAvidin and dye coupling to a double-cysteine mutant of cowpea mosaic virus (CPMV) were found to be comparable to the commonly used streptavidin-phycoerythrin (SAPE) for high density resequencing microarray. A 3-fold signal enhancement in comparison to Cy5-dCTP controls was obtained when using nanoparticles on control scorecard expression microarrays. Hybridization results from commercially available 8000 rat expression arrays indicate an increment of 14% on the detected features when the virus complex was used as the staining reagent in comparison to Cy5-dCTP controls. The current work shows the utility of the CPMV-dye nanoparticles as a detection reagent in well-established detection platforms. 相似文献
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Fouqué B Brachet AG Gétin S Pégon P Obeïd P Delapierre G Chatelain F 《Biosensors & bioelectronics》2007,22(9-10):2151-2157
Today, microarray fluorescence detection is still limited because a great proportion of hybrids remain undetectable. In this paper we describe sol-gel optical multilayers (stacks of low- and high-index layers) deposited on glass slides which increase the fluorescence of DNA microarrays and favour the detection of fluorescent targets. An alternative to the expensive and time-consuming physical vapour deposition technology is proposed. It is a low-cost sol-gel coating of glass slides, each layer being made by "dipping" (alternatively in SiO2 or TiO2 solutions), "draining and drying". After the selection of the best surface layer of the substrates, the multilayer mirrors modelled for one (Cy3) or two (Cy3 and Cy5) fluorophores are spotted with a series of Yeast probes and compared to similar microarrays on standard glass slides through hybridisation experiments. The fluorescence images of the mirrors show increased signals for all the probes. The enhancement factors determined for Cy3 and for Cy3/Cy5 mirrors (10-12 and 4-5, respectively) are consistent with the initial modelling. This allows the assessment of the basal expression levels of Yeast low-expressed genes. Moreover, these substrates show a noticeable increase in sensitivity for induction/repression ratio measurements in differential gene expression experiments. So, they could be considered as promising tools for the analysis of small biological samples. 相似文献
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Genetic analysis of high-risk e6 in episomal maintenance of human papillomavirus genomes in primary human keratinocytes 总被引:1,自引:0,他引:1
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Papillomaviruses possess small DNA genomes that encode five early (E) proteins. Transient DNA replication requires activities of the E1 and E2 proteins and a DNA segment containing their binding sites. The E6 and E7 proteins of cancer-associated human papillomavirus (HPV) transform cells in culture. Recent reports have shown that E6 and E7 are necessary for episomal maintenance of HPV in primary keratinocytes. The functions of E6 necessary for viral replication have not been determined, and to address this question we used a recently developed transfection system based on HPV31. To utilize a series of HPV16 E6 mutations, HPV31 E6 was replaced by its HPV16 counterpart. This chimeric genome was competent for both transient and stable replication in keratinocytes. Four HPV16 E6 mutations that do not stimulate p53 degradation were unable to support stable viral replication, suggesting this activity may be necessary for episomal maintenance. E7 has also been shown to be essential for episomal maintenance of the HPV31 genome. A point mutation in the Rb binding motif of HPV E7 has been reported to render HPV31 unable to stably replicate. Interestingly, HPV31 genomes harboring two of the three p53 degradation-defective E6 mutations combined with this E7 mutation were maintained as replicating episomes. These findings imply that the balance between E6 and E7 functions in infected cells is critical for episomal maintenance of high-risk HPV genomes. This model will be useful to dissect the activities of E6 and E7 necessary for viral DNA replication. 相似文献
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Increasing the sensitivity of DNA microarrays by metal-enhanced fluorescence using surface-bound silver nanoparticles 总被引:1,自引:0,他引:1
The effects of metal-enhanced fluorescence (MEF) have been measured for two dyes commonly used in DNA microarrays, Cy3 and Cy5. Silver island films (SIFs) grown on glass microscope slides were used as substrates for MEF DNA arrays. We examined MEF by spotting biotinylated, singly-labeled 23 bp DNAs onto avidin-coated SIF substrates. The fluorescence enhancement was found to be dependent on the DNA spotting concentration: below ~12.5 μM, MEF increased linearly, and at higher concentrations MEF remained at a constant maximum of 28-fold for Cy5 and 4-fold for Cy3, compared to avidin-coated glass substrates. Hybridization of singly-labeled oligonucleotides to arrayed single-stranded probes showed lower maximal MEF factors of 10-fold for Cy5 and 2.5-fold for Cy3, because of the smaller amount of immobilized fluorophores as a result of reduced surface hybridization efficiencies. We discuss how MEF can be used to increase the sensitivity of DNA arrays, especially for far red emitting fluorophores like Cy5, without significantly altering current microarray protocols. 相似文献