基因芯片技术检测3种肠道病原微生物方法的建立

目的:建立一种运用多重PCR和基因芯片技术检测和鉴定伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的方法。方法:分别选取伤寒沙门氏菌染色体ViaB区域中编码调控Vi抗原表达的基因(vipR)、痢疾杆菌编码侵袭质粒抗原H基因(ipaH)和单核细胞增生利斯特菌溶血素基因(hlyA)设计引物和探针,探针3'端进行氨基修饰,下游引物标记荧光素Cy3。在优化的PCR和杂交反应条件下,进行三重PCR扩增,产物与包括3种致病菌特异性探针的基因芯片杂交。在评价基因芯片的特异性和灵敏度之后,对临床样本进行检测。结果:只有3种目的致病菌的PCR产物在相应探针位置出现特异性信号,其他阴性细菌均无信号出现;3种致病菌的检测灵敏度均可达到103CFU/mL;检测30例临床样本的结果与常规细菌学培养结果一致。结论:所建立的可同时检测伤寒沙门氏菌、痢疾杆菌和单核细胞增生利斯特菌的基因芯片方法快速、准确,特异性高,重复性好,为3种肠道致病菌的快速检测和鉴定提供了新方法和新思路。     

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

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     

利用基因芯片技术区分禽流感病毒主要亚型

[目的]研制可同时区分AIV的H5、H7、H9血凝素亚型及N1、N2神经氨酸酶亚型的基因诊断芯片.[方法]分别克隆了禽流感病毒的M基因,H5、H7、H9亚型HA基因,N1、N2亚型NA基因以及看家基因GAPDH的重组质粒.以重组质粒为模板,用PCR方法扩增制备探针,纯化后点于氨基修饰的片基上,制备基因芯片.在PCR过程中对待检样品进行标记,然后与芯片杂交,洗涤,扫描并进行结果分析.[结果]结果显示检测探针可特异性的与相应的标记样品进行杂交,呈现较强的杂交信号,且无交叉杂交.同时用RT-PCR、鸡胚接种和基因芯片方法对H1-H15亚型AIV参考毒株、30份人工感染样品、21份现地疑似样品进行检测,结果发现,对人工感染样品芯片检测方法与鸡胚接种和RT-PCR的符合率分别为100%和96%,现地样品符合率为100%.[结论]研究表明该方法可用于同步鉴别部分主要流行的禽流感亚型,是一种有效的新方法.     

Quantification of mRNA Levels by Fluorescently Labelled Reversde Transcription Competitive PCR

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Genome-wide analysis of lipoprotein expression in Escherichia coli MG1655

To gain insight into the cell envelope of Escherichia coli grown under aerobic and anaerobic conditions, lipoproteins were examined by using functional genomics. The mRNA expression levels of each of these genes under three growth conditions--aerobic, anaerobic, and anaerobic with nitrate--were examined by using both Affymetrix GeneChip E. coli antisense genome arrays and real-time PCR (RT-PCR). Many genes showed significant changes in expression level. The RT-PCR results were in very good agreement with the microarray data. The results of this study represent the first insights into the possible roles of unknown lipoprotein genes and broaden our understanding of the composition of the cell envelope under different environmental conditions. Additionally, these data serve as a test set for the refinement of high-throughput bioinformatic and global gene expression methods.     

RT-PCR检测HUTP14A mRNA时排除假基因HUTP14C干扰的方法

人类U3蛋白14C基因(HUTP14C)是人类U3蛋白14A基因(HUTP14A)的假基因。两者转录本序列同源性高达95%。常规RT-qPCR技术在检测HUTP14A mRNA丰度时,HUTP14C的存在会影响检测结果。本研究旨在建立检测HUTP14A mRNA时排除HUTP14C干扰的RT PCR方法。本研究设计出能分别从多种肿瘤细胞DNA和RNA中特异性扩增HUTP14A和HUTP14C的引物,避免假基因HUTP14C对其同源基因HUTP14A检测的干扰。在检测细胞系HUTP14A mRNA时,通过DNaseⅠ消除RNA中污染的HUTP14C DNA,用靶向HUTP14C 3′-UTR的siRNA沉默HUTP14C mRNA后,再用RT PCR检测HUTP14A mRNA丰度,使结果更加准确。在18对肝癌及癌旁组织中,利用特异性引物进行RT PCR检测,HUTP14A和HUTP14C mRNA的表达略高于癌旁组织。本研究提示,针对有假基因存在的功能基因,对其mRNA丰度进行检测时,在提取细胞或组织总RNA后,用DNaseⅠ处理,再用RNA直接进行PCR扩增,排除DNA污染后,再进行RT-PCR或RT-qPCR扩增。大多假基因具有较长的3′-UTR区,在该区域设计siRNA特异性沉默假基因的mRNA后,用RT-qPCR检测功能基因的mRNA丰度,可以排除假基因mRNA的影响。在病理组织中检测功能基因的mRNA丰度时,可以根据假基因和其功能基因的序列差异设计出特异扩增功能基因的引物,从假基因的3′-UTR区设计特异扩增假基因的引物,通过RT-qPCR技术分别检测二者的mRNA。     

Bax基因诱导鼻咽癌细胞中相关基因的mRNA差异显示分析

目的：应用差异显示技术,筛选鼻咽癌相关基因的异常表达。方法：选用连接有bax基因的真核表达质粒pSFFV-bax-neo, 采用脂质体转染法, 将其转染到CNE2细胞中, 以转染有空载的pSFFV-neo 质粒的CNE2细胞为对照, 采用Trizol试剂快速抽取法, 提取mRNA经逆转录成cDNA,用锚式引物和随机引物进行PCR扩增, 加入同位素, 用6%的测序变性聚丙烯酰胺凝胶上电泳PCR产物进行分离。切下聚丙烯酰胺凝胶上明显的6条差异带, 回收差异cDNA, 进行第二次PCR扩增, 经低溶点琼脂凝胶回收, 获取大量PCR扩增的差异cDNA片段,同位素标记作为探针, 抽取RNA, 进行点样, 杂交, 洗膜放射自显影等步骤, 进行细胞RNA的Northern印迹斑点杂交。结果：表明4条cDNA片段均为CNE2细胞表达片段。结论：发现bax可诱导CNE2细胞中有某些相关基因表达,并抑制了CNE2细胞某些相关基因表达。     

Simple and effective method for generating single-stranded DNA targets and probes

A simple and efficient PCR method was developed for generating dye- or radiolabeled single-stranded DNA targets or probes used for hybridization studies. The method involved the use of a pair of long primers with high annealing temperatures and a short, labeled primer with a low annealing temperature in a PCR consisting of two cycles at different temperatures. We used this method to generate dye Cy 5-labeled and [32P]-radiolabeled single-stranded DNA targets and probes. These labeled probes were used successfully for the microarray identification of point mutations in Mycobacterium tuberculosis genes and for the Northern blot detection of expression changes of the GATA-2 gene in Pneumocystis carinii-infected rat lungs.     

How is mRNA expression predictive for protein expression? A correlation study on human circulating monocytes

A key assumption in studying mRNA expression is that it is informative in the prediction of protein expression. However, only limited studies have explored the mRNA-protein expression correlation in yeast or human tissues and the results have been relatively inconsistent. We carried out correlation analyses on mRNA-protein expressions in freshly isolated human circulating monocytes from 30 unrelated women. The expressed proteins for 71 genes were quantified and identified by 2-D electrophoresis coupled with mass spectrometry. The corresponding mRNA expressions were quantified by Affymetrix gene chips. Significant correlation ( r =0.235, P <0.0001) was observed for the whole dataset including all studied genes and all samples. The correlations varied in different biological categories of gene ontology. For example, the highest correlation was achieved for genes of the extracellular region in terms of cellular component ( r =0.643, P <0.0001) and the lowest correlation was obtained for genes of regulation ( r =0.099, P=0.213) in terms of biological process. In the genome, half of the samples showed significant positive correlation for the 71 genes and significant correlation was found between the average mRNA and the average protein expression levels in all samples ( r =0.296, P <0.01). However, at the study group level, only five studied genes had significant positive correlation across all the samples. Our results showed an overall positive correlation between mRNA and protein expression levels. However, the moderate and varied correlations suggest that mRNA expression might be sometimes useful, but certainly far from perfect, in predicting protein expression levels.     

半定量RT-PCR检测运动发酵单胞菌中外源基因转录水平的研究

本研究运用半定量RT-PCR法检测运动发酵单胞菌重组菌中外源基因xylB的转录水平。提取野生型运动发酵单胞菌CP4及其2个重组菌的总RNA, 检测无DNA污染后定量至同一浓度、并反转录为cDNA。观测目的基因xylB和内标基因16S rRNA的PCR扩增曲线、并确定合适的循环数, 选用相同量的cDNA为模板, PCR检测各样本中xylB相对16S rRNA的转录水平。结果表明野生型菌株CP4中xylB基因没有转录, 而两株重组菌中皆有xylB的转录本, 且转录丰度基本一致, 酶活测定也进一步证实该基因在重组菌中有效表达。该方法可用于鉴定运动发酵单胞菌中特定基因的转录水平, 是一种快速有效的检测方法。     

Design and application of an oligonucleotide microarray for the investigation of compost microbial communities

A microarray consisting of oligonucleotide probes targeting variable regions of the 16S rRNA gene was designed and tested for the investigation of microbial communities in compost. Probes were designed for microorganisms that have been previously reported in the composting process and for plant, animal and human pathogens. The oligonucleotide probes were between 17 and 25 bp in length and included mostly species-specific sequences. Validation of probe specificity and optimization of hybridization conditions were conducted using fluorescently labeled 16S rRNA gene PCR products of pure culture strains. A labeling method employing a Cy3 or Cy5-labeled forward primer together with a phosphate-conjugated reverse primer for the production of single stranded DNA after a digestion step was optimised and used to label target DNA. A combination of two different DNA extraction methods using both physical and chemical lysis was found to give the best DNA yields. Increased hybridization signal intensities were obtained for probes modified with a 12 mer T-spacer. The microarray was found to have a detection limit of 10(3) cells, although in compost spiking experiments, the detection limit was reduced to 10(5) cells. The application of the microarray to compost samples indicated the presence of Streptococcus, Acinetobacter lwoffii, and Clostridium tetani in various compost samples. The presence of A. lwoffii in those compost samples was confirmed by PCR using primers specific for the organism. The aim of this study was to develop a molecular tool that would allow screening for the presence or absence of different microorganisms within compost samples.