Amplification-free detection of grapevine viruses using an oligonucleotide microarray

A single-colour microarray hybridization system was designed and evaluated for the detection of viruses infecting grapevine. Total RNA (≥0.5μg) from infected plants was converted to cDNA and labelled with Cy3 using two different strategies. While amine-modified and labelled cDNA was adequate for the detection of nepoviruses, the 3DNA technique, a post-hybridization detection method that uses intensely fluorescent dendrimer reagents, was required for the detection of closteroviruses in infected plants. Threshold detection levels were based on the ratio between viral specific and 18S rRNA positive control signal intensities. Oligonucleotides between 27 and 75 nucleotides in length were evaluated and compared. Viruses detected include eight nepoviruses, two vitiviruses, and one each of closterovirus, foveavirus, ampelovirus, maculavirus and sadwavirus. Results of this work demonstrate the potential of microarray technique to detect viral pathogens without sequence bias amplification of template RNA.     

Microarray analysis using amplified mRNA from laser capture microdissection of microscopic hepatocellular precancerous lesions and frozen hepatocellular carcinomas reveals unique and consistent gene expression profiles

The indirect labeling cDNA microarray technique was used to evaluate gene expression profiles of pure cell populations from frozen sections of carcinomas and adenomas harvested from precancerous hepatocellular lesions by using laser capture microdissection (LCM). The levels of differentially expressed genes were investigated using a cDNA microarray with 9,984 features with only 2 ug of two-round amplified aRNA, equivalent to 35 cells from LCM-adenomas and frozen samples of carcinomas from simian virus 40 (SV40) large T antigen transgenic rats. A total of 855 genes were identified as being 3-fold or more differentially expressed in carcinomas or adenomas as compared to normal tissue controls. Among these 855 genes, 71 genes were differentially expressed in both carcinomas and adenomas. Commonly up-regulated genes in both carcinoma and adenomas were 28 while 41 of the 71 genes were commonly down-regulated. Two genes, Igh1 (immunoglobulin heavy chain 1(Serum IgG2a), Image clone ID: 875880) and EST clone (AI893585, Image clone ID: 596604) were more than 7-fold up-regulated in carcinomas and 6-fold down-regulated in adenomas. In Cy5 and Cy3 reciprocal experiments for screening out false positive signals, the amplified carcinomas showed higher Pearson Correlation Coefficient values (-0.94 and -0.92) than the LCM-amplified adenoma samples (-0.79 and -0.84). LCM-amplified samples provided higher signal intensities over backgrounds and a greater average of Cy5:Cy3 ratios. Expression levels of mRNAs from selected genes, determined by using traditional dot blot analysis, revealed that 36 of 40 tested expression profiles were consistent with the microarray data. Thus, amplified aRNA harvested from homogeneous cell types using LCM can be applied to study gene expression profiles by use of microarray analysis.     

Lovastatin对肝癌HepG2细胞作用后基因差异表达的初步研究

目的： 用cDNA芯片观察lovastatin作用前后HepG2细胞的差异表达基因。 方法： 20 μmol/L的lovastatin作用于HepG2细胞18 h，各提取实验组与对照组细胞总RNA，用cDNA直接标记法制备探针，进行杂交，杂交信号经扫描后，用软件分析基因表达情况。应用RT-PCR验证部分差异表达基因。 结果： Lovastatin作用后表达上调的基因有30个，表达下调的基因有11个，涉及信号转导、肿瘤免疫、细胞周期等方面，RT-PCR结果符合基因芯片结果。 结论： 用基因芯片筛选出的lovastatin作用后的肝癌细胞差异表达基因为深入研究他汀类药物的抗肿瘤机制提供重要的理论依据。     

胰腺癌相关基因寡核苷酸芯片的制备和初步应用(英文)

目的：研究胰腺癌相关基因寡核苷酸芯片的构建及其在检测胰腺癌基因表达谱方面的应用。方法：有目的地筛选胰腺癌相关基因，采用合成后点样的方法将合成的寡核苷酸探针点于同型双功能偶联剂（APS-PDC）包被的基片上，制成寡核苷酸基因芯片。Trizol法抽提组织总RNA，并用Qiagen Rneasy kit 进一步纯化。在cDNA第1链合成过程中，通过反转录酶将CyDye标记核苷酸直接渗入到cDNA中制备荧光探针，其中用Cy3-dCTP标记胰腺癌组织，Cy5-dCTP标记正常胰腺组织。将荧光标记探针定量后与芯片杂交16-18 h。用Agilent 扫描仪进行扫描，Imagene3.0软件（Biodiscovery,Inc.）图像分析，计算2种荧光Cy3 与Cy5信号强度的比值。挑选差异基因CDC25B和TUSC3进行荧光定量PUR（Sybrgreen方法）验证，以B-actin基因为内参照基因，PCR产物的定量方法采用比较Ct法。结果：芯片杂交扫描图像信号清晰，具有较低的整体背景和较高的信噪比，各阳性质控点信号均匀一致，阴性质控点和空白点信号低。与正常组织相比，胰腺癌组织中差异表达基因24条，占全部基因的25.5%，其中上调基因17条（占18.1%），下调基因7条（占7.4%）。荧光定量PCR验证，CDC25B和TUSC3基因在胰腺癌中的表达分别为上调和下调，其表达趋势与芯片实验的结果一致。结论：制备的胰腺癌相关基因芯片，可同时、并行检测多种胰腺癌相关基因的表达改变，具有一定的特异性和灵敏性。胰腺癌组织与正常胰腺组织相比，基因表达谱具有明显的差异，为进一步研究胰腺癌的发病机理和早期诊断提供了有用的信息。     

Easy and fast detection and genotyping of high-risk human papillomavirus by dedicated DNA microarrays

Persistent cervical high-risk human papillomavirus (HPV) infection is correlated with an increased risk of developing a high-grade cervical intraepithelial lesion. A two-step method was developed for detection and genotyping of high-risk HPV. DNA was firstly amplified by asymmetrical PCR in the presence of Cy3-labelled primers and dUTP. Labelled DNA was then genotyped using DNA microarray hybridization. The current study evaluated the technical efficacy of laboratory-designed HPV DNA microarrays for high-risk HPV genotyping on 57 malignant and non-malignant cervical smears. The approach was evaluated for a broad range of cytological samples: high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of high-grade (ASC-H). High-risk HPV was also detected in six atypical squamous cells of undetermined significance (ASC-US) samples; among them only one cervical specimen was found uninfected, associated with no histological lesion. The HPV oligonucleotide DNA microarray genotyping detected 36 infections with a single high-risk HPV type and 5 multiple infections with several high-risk types. Taken together, these results demonstrate the sensitivity and specificity of the HPV DNA microarray approach. This approach could improve clinical management of patients with cervical cytological abnormalities.     

Development of a DNA microarray for simultaneous detection and genotyping of lyssaviruses

The lyssavirus genus of the Rhabdoviridae family of viruses includes 7 genotypes and several non-assigned isolates. The source of lyssavirus infections is diverse with numerous reservoirs in a wide geographical area. In many parts of the world reservoir hosts can potentially be carrying one of several lyssavirus strains and possibly new divergent isolates await discovery. Accordingly, generic detection methods are required to be able to detect and discriminate all lyssaviruses and identify new divergent isolates. Here we have allied a sequence-independent amplification method to microarray to enable simultaneous detection and identification of all lyssavirus genotypes. To do so, lyssavirus RNA was converted to cDNA and amplified in a random PCR, labelled and hybridized to probes on the microarray chip before being statistically analysed. The probes were to a 405 bp region of the relatively conserved N gene. Here we demonstrate a microarray capable of detecting each of the seven lyssavirus genotypes. The random amplification of lyssavirus RNA and the numerous oligonucleotide probes on the microarray chip also offer the potential to detect novel lyssaviruses.     

寡聚核苷酸微阵列技术应用于脉动流剪应力作用下人体动脉平滑肌细胞基因差异表达的研究

采用本实验室自行设计和研制的 Flow Cham ber血液流动剪切装置 ,从细胞生物力学的角度 ,采用寡聚核苷酸微阵列技术 ,探讨人体脐动脉平滑肌细胞 (VSMC)在脉动流剪应力作用下的基因差异性表达 ,以及相关基因表达的调控 ;比较脉动流和定常流两种剪应力作用下 VSMC基因表达的差异。通过提取细胞总 RNA ,逆转录合成单链 c DNA,双链 c DNA,体外转录合成生物素标记的 c DNA与基因进行芯片杂交 ,经抗体的检测标记荧光染料Cy3,用基因芯片扫描仪进行基因芯片的图像扫描。结果发现 ,与对照组比较 ,经脉动流剪应力作用后 ,血管平滑肌细胞有 1330个基因出现差异表达 ;经定常流剪应力作用后 ,与对照组比较 ,有 2 6 76个基因出现差异表达 ;比较定常流和脉动流两组的血管平滑肌细胞基因 ,有 2 2 97个基因存在表达差异。实验表明 ,近生理状态的脉动流与定常流两种剪应力 ,对体外培养的人体血管平滑肌细胞的基因表达 ,在数量、范围和调控水平上有着很大的差异。提示在生理或病理情况下 ,血液流体力学的变化 ,可诱导血管平滑肌细胞基因在 m RNA表达水平出现不同的应力响应。     

The effects of different sample labelling methods on signal intensities of a 60-mer diagnostic microarray

The effects of four different labelling methods on signal intensities of a 60-mer diagnostic microarray were studied. Eighty of virus-specific oligonucleotide probes for human influenza virus were prepared in an array of 15x16 spots. RNA samples from cultured human influenza virus strains were labelled with four different methods, including direct cDNA labelling (DL), universal primer labelling (UPL), direct cDNA labelling with restriction display (DL-RD), and Cy-dUTP incorporated cDNA labelling with restriction display (IL-RD) in a signal color format. The background-subtracted signal intensities from five replicate hybridization experiments of each labelling method were analyzed using one-way analysis of variance (one-way ANOVA) and linear regression techniques. The effect of sample labelling method on background-subtracted signal intensities was significant (p<0.001) and multiple comparisons showed the differences existed mainly between DL and the other three labelling methods. The sample labelling method explained about 4.3% of signal intensity. The results demonstrated that UPL and the RD-based methods are more efficient than the conventional DL method for sample labelling, an important variation factor affecting the signal intensities in diagnostic microarrays.     

流感/禽流感病毒及其致病力鉴别的基因芯片技术研究

目的 建立流感/禽流感病毒及其致病力鉴别的基因芯片检测技术.方法 以血凝素(HA)、神经氨酸酶(NA)、核蛋白(NP)基冈作为靶片段,设计病毒检测和致病力特异性鉴别探针,建立基因芯片鉴别检测技术,采用单引物扩增法(SPA)处理样本核酸,分别对此芯片进行特异性、敏感性和符合率评价.结果 此芯片能够特异性的检测H1N1、H3N2、B型流感病毒及H5N1、H9N2禽流感病毒,敏感性分别为8HAU、16HAU、32HAU及8HAU、8HAU.致病力鉴别探针敏感性为32HAU.同RT-PCR方法比较,检测灵敏度为83.9%.结论 建立的常见流感病毒检测基因芯片特异性高、敏感性高、灵敏度高,更能够对致病力进行有效甄别,可作为临床诊断、传染病防控等方面的有益补充.     

线粒体多态性检测寡核苷酸芯片的构建

目的构建用于线粒体DNA（mtDNA）D-环控制区（D—LOOP区）多态性多个位点集成检测的寡核苷酸芯片。方法根据GenBank中人mtDNA D—LOOP区序列设计2组引物[普通引物和N,N＇-对羧苄基吲哚三菁（Cy5）标记引物]和55种寡核苷酸探针。将探针固定于醛基修饰载玻片表面,制备mtDNA多态性检测芯片。提取40例健康人外周血标本mtDNA,应用Cy5标记引物不对称PCR扩增mtDNA D—LOOP区片段。取未变性和变性后扩增产物分别与芯片进行杂交,观察二者杂交信号强度差异,并将芯片检测结果与DNA测序结果进行比较。观察等倍稀释的不对称PCR扩增产物的杂交信号强度,检测芯片的灵敏度;将DNA测序结果与探针进行BLAST2比对,找出与PCR扩增产物完全匹配和仅1个碱基不匹配的探针,分析二者在芯片上相应位点杂交信号强度的差异,观察芯片的特异性;以放置6个月后的芯片检测外周血mtDNA,观察杂交信号强度的变化。结果不对称PCR扩增获得的mtDNA D—LOOP区片段（466bp）与预期表达片段大小一致。不对称PCR扩增产物直接杂交和变性后杂交的信号强度无明显差异,40例健康人外周血标本mtDNA的芯片杂交检测结果均与DNA测序结果完全吻合。灵敏度检测结果显示,杂交信号强度随扩增产物稀释程度的增加而减弱,芯片检测靶分子的灵敏度为0．1ng;与DNA测序结果完全匹配的探针174和仅相差1个碱基“C”的探针174c的杂交信号强度具有明显差异;而保存6个月后的芯片杂交信号强度基本保持不变。结论构建的寡核苷酸芯片可满足对mtDNA D—LOOP区多态性进行多个位点快速通量检测的需要,具有较高的灵敏度和特异性,适用于法医鉴定及线粒体疾病的检测。     

A modified restriction display PCR method in sample-labelling of DNA microarray

The restriction display PCR is a useful technique for studying the diversity of gene expression. This method involves ligating the digested genes with adapters and amplifying the gene fragments by PCR using universal and selective primers. In this study, we improved this restriction display PCR method by using Cy3-UP, a fluorescently labelled universal primer, in place of Cy3-dCTP in sample-labelling for DNA microarray. The results show that this new method increases significantly the sensitivity of the assay, and will have a wide application in the DNA microarray field.     

Plant virus cDNA chip hybridization for detection and differentiation of four cucurbit-infecting Tobamoviruses

A plant virus cDNA chip was developed by using viral cDNA clones and microarray technology. The cDNA chip was designed for detection and differentiation of the four species of selected cucurbit-infecting tobamoviruses [target viruses: Cucumber green mottle mosaic virus (CGMMV); Cucumber fruit mottle mosaic virus (CFMMV); Kyuri green mottle mosaic virus (KGMMV); and Zucchini green mottle mosaic virus (ZGMMV)]. The chip consisted of cDNA clones of the four cucurbit-infecting tobamoviruses, two target-related tobamoviruses, and another three unrelated plant viruses. Polymerase chain reaction products were amplified from the selected cDNA clones and arrayed onto slide glass. The cDNA chip, which was called cucurbit-virus chip, detected successfully specific target viruses. When applied to probes made from ZGMMV-infected samples, ZGMMV reacted strongly with its homologous cDNA and moderately reacted with KGMMV and CFMMV, while it did not react with CGMMV on the same chip. CGMMV probe gave strong signal intensity to its homologous cDNA spot and weakly reacted with ZGMMV, KGMMV, and CFMMV. The signal intensity of all combinations of probe and target was correlated significantly with nucleotide sequence identities between the probes and target viruses based on scatter diagrams. The signals could be made as image files for specific virus detection, and this could be useful for virus identification and differentiation. This is the first report of plant virus detection by using cDNA chip technology.     

常染色体显性多囊肾组织差异表达基因的初步研究

目的应用基因芯片技术及最新公共数据库，筛选常染色体显性多囊肾组织中差异表达的基因，对其进行功能分类，并对其中1条基因利用原位杂交技术进行验证。方法将代表8398条人类基因的PCR产物制成基因芯片。将等量的多囊肾组织和正常肾组织mRNA分别用Cy5、Cy3荧光标记，逆转录合成cDNA探针，混合后与上述基因芯片杂交。扫描杂交信号荧光强度，找出差异表达基因，对获得的基因进行分子生物信息学分析。并对其中的上调表达基因IGF1 mRNA进行原位杂交，验证基因芯片结果的准确性。结果（1）在进入研究的8398条基因中，共发现357条差异表达基因。94条基因在多囊肾组织中低表达，263条基因高表达；（2）上调表达基因主要属于原癌基因，细胞骨架蛋白和运动相关蛋白，凋亡相关蛋白，细胞信号和传递蛋白，细胞因子；下调表达基因主要属于抑癌基因，DNA结合、转录和转录因子，细胞信号和传递蛋白，参与代谢的基因；（3）IGF1 mRNA原位杂交结果与芯片结果一致。结论基因表达谱芯片可快速、高效地筛选差异表达基因；多囊肾病的发生、发展中存在着多种不同功能基因表达调控的改变。     

Enhancing sensitivity of human herpes virus diagnosis with DNA microarrays using dendrimers

DNA microarray technology has become a promising new tool for the detection and identification of viral pathogens in human plasma and cell cultures. For exploration of this technology, we have developed DNA microarrays that encode capture oligonucleotide probes for different human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The on-chip hybridization is accomplished with the PCR amplicons of the respective human herpes virus types. In this original article, we attached multiple Cy3-fluorophores to the branched 5' ends of the labeling oligonucleotide primers. For the first time, we experimentally demonstrated that the self-designed, knowledge-based, and focused microarrays specifically hybridized to fluorophore-labeled pathogenic DNAs using dendrimer technology. The fluorescence signal enhancement via the dendrimers was up to 30 times compared with the quenched single Cy3-fluorophore-labeled HSV-1 DNA. The on-chip signal-amplifying effect depended upon the number of branches and the concentration of fluorophore-labeled pathogenic DNAs. Treblers were superior to doublers, as trebler-labeled nucleic acids had fluorescence-signal-enhancing effects over a broad range of labeled DNA concentrations exemplified for the quenched single Cy3-fluorophore-labeled HSV-1 and non-quenched single Cy3-fluorophore-labeled CMV DNAs.     

引物标记与掺入标记在HBV基因多态性芯片检测中应用的研究

目的 研究引物标记与掺入标记在乙型肝炎病毒（HBV）基因多态性芯片检测中的应用。方法 用掺入标记或引物标记荧光分子Cy5，扩增HBV P区、前C／C区，制备含荧光分子Cy5的目的片段后，分别与HBV基因多态性芯片杂交、扫描并分析结果。结果 掺入标记法信号强度略高于引物标记法，但非特异信号强度也高，两法检测42份乙型肝炎患者血清，结果完全一致，重复性均较好，批内CV15％－20％，引物标记法用于检测HBV基因多态性灵敏度达10^4 copies/ml。结论 引物标记法信号强度虽略低于掺入标记法，但检测灵敏度、特异性仍满足临床要求。     

Resequencing microarray for detection of human adenoviruses in patients with conjunctivitis

BackgroundAlthough high-density resequencing microarray is useful for detection and tracking the evolution of viruses associated with respiratory tract infections, no report on using this technology for the detection of viruses in patients with conjunctivitis is available.ObjectivesTo test if high-density resequencing microarray can be applied to detection of viruses in conjunctival swabs for patients with conjunctivitis.Study designIn this prospective proof-of-concept study, every 4 or 5 bacterial culture-negative conjunctival swab samples were pooled and subject to viral detection using TessArray? Resequencing Pathogen Microarrays-Flu 3.1 (RPM-Flu-3.1). Results were compared with human adenovirus (HAdV) hexon gene PCR sequencing and viral culture.ResultsThirty-two of the 38 conjunctival swab samples were bacterial culture-negative. Four of the 7 pooled samples were positive for HAdV using RPM-Flu-3.1. Hexon gene PCR sequencing on the 38 original individual samples showed that 3 and 4 samples contained HAdVs species D and B respectively. All the 6 samples that were positive for hexon gene PCR but negative for bacterial culture were also positive by the resequencing microarray. Viral culture was positive for HAdV type 3 in 1 sample, which was also positive by PCR and resequencing microarray.ConclusionsResequencing microarray is as sensitive as PCR for detection of HAdV in conjunctival swabs. Unlike viral culture and hexon gene PCR sequencing, resequencing microarray was not able to differentiate the type and species of HAdV. Development of microarrays for conjunctivitis can be performed for rapid diagnosis of the viral cause of conjunctivitis.     

Fabrication of oligonucleotide microarray for the detection of Japanese encephalitis virus

A low-density oligonucleotide microarray was used for the detection of Japanese encephalitis virus (JEV) , combining with restriction display PCR labeling method. The hybridization targets were amplified from 6 plasmids containing several JEV gene fragments. Corresponding oligonucleotide probe spots were detected unambiguously. We claim that the oligonucleotide microarray technology is feasible and may have potential for clinical laboratory application.