一种提高基因芯片检测中杂交信号强度的简单方法

目的 改进PCR产物和固定探针杂交效率。提高信噪比，以提高基因芯片检测的灵敏度。方法 以淋球菌隐蔽型质粒pJD1的基因芯片检测为例，在荧光素标记引物的PCR产物中加入不同浓度的Taq酶抑制剂EDTA及位于固定探针所在位点的两翼的荧光素标记探针，一起变性后再进行常规杂交、冲洗。结果 加入EDTA和荧光素标记探针使基因芯片检测的荧光强度能提高6倍以上。结论 加入EDTA和荧光素标记探针是一种提高基因芯片检测的效率的简单而有效的方法。     

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。     

微量核酸样品的双重限制性荧光标记技术

目的 采用双重限制性荧光标记技术(double restriction fluorescent labeling,DRFL),标记微量核酸样品,探索提高基因芯片用于病原体检测的灵敏度.方法 以限制性显示技术处理SARS-CoV核酸样品,分别采用传统限制性荧光标记技术(直接采用荧光标记的通用引物标记)和双重限制性荧光标记技术(荧光标记的通用引物和荧光标记的dNTP的双重荧光标记)进行处理,并进一步与基因芯片进行杂交,在同等条件下进行杂交、清洗和进行基因芯片的扫描检测.通过对杂交结果的分析,比较两种标记方法的标记效果.结果 以DRFL方法标记SARS基因片段,其荧光强度均值提高了3.5835倍,但互相对应的每个探针之间的荧光信号提高程度存在差异.结论 DRFL技术能有效地提高单位分子标记片段的荧光强度值,并进一步提高了检测的灵敏度,可用于微量病原体核酸样品的基因芯片检测.     

HIV-1G亚型基因芯片的制备与杂交分析

基因芯片技术是一种高效、敏感、平行化的基因检测分析新技术[1],对临床疾病的诊断技术将产生革命性的影响,目前主要集中在感染性病原基因的检测方面。我们尝试将该技术应用于人免疫缺陷病毒(HIV)基因的检测与分型。首先制备HIV基因组探针并分别进行分析,实验中我们采用一种基因显示新技术:限制性显示(restriction display,RD-PCR)[2、3],制备HIV-1G亚型基因探针,并用基因芯片荧光标记杂交的方法对这些探针进行了杂交分析,为基因芯片技术在HIV感染检测的应用中奠定了基础。     

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。     

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。     

高性价比寡核苷酸基因表达谱芯片的制备方法

目的探讨探针长度对寡核苷酸(Oligo)基因芯片杂交信号的影响。方法以大肠杆菌基因表达信息作为实验模型,根据已有大肠杆菌全基因组芯片数据选择覆盖高、中、低杂交信号强度的20个大肠杆菌基因,针对该20个基因分别设计59-mer和70-mer长度Oligo探针,并将2种探针点制在同一张芯片中,同时设阳性对照探针和阴性对照点样液。提取大肠杆菌RNA,经过反转录、扩增、荧光标记后与芯片进行杂交反应,采用激光共聚焦扫描仪扫描芯片,利用数据分析软件提取探针的杂交信号值并进行显著性分析。结果大肠杆菌28SrRNA和18SrRNA条带清晰,无降解带出现,质量合格。芯片杂交结果显示59-mer和70-mer长度探针的杂交效率和杂交信号差异无统计学意义(P=0.9810),阳性对照探针出现阳性杂交信号,阴性对照点样液未检测到杂交信号,符合质控要求。结论59-mer长度探针可用来制备Oligo基因芯片,这不仅降低了基因芯片制作成本,而且将推动基因芯片技术更为普及的应用。     

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-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.     

微量核酸样品的双重限制性荧光标记技术

目的采用双重限制性荧光标记技术(doub le restriction fluorescent labeling,DRFL)标记微量核酸样品,提高基因芯片用于病原体检测的灵敏度。方法以限制性显示技术处理SARS-CoV核酸样品,分别采用传统限制性荧光标记技术(直接采用荧光标记的通用引物标记)和双重限制性荧光标记技术(荧光标记的通用引物和荧光标记的dNTP的双重荧光标记)标记,进一步在同等条件下与基因芯片进行杂交、清洗和芯片扫描检测。通过对杂交结果的分析,比较2种标记方法的标记效果。结果以DRFL方法标记SARS基因片段,其荧光强度均值比传统荧光标记方法提高了3.5835倍。结论DRFL技术有效地提高单位分子标记片段的荧光强度值,提高了检测的灵敏度,可用于微量病原体核酸样品的基因芯片检测。     

交联型丙烯酸酯聚合物分子层用于固定寡核苷酸分子初探

目的探讨在显微玻片上衍生聚合物分子层用于固定寡核苷酸分子的方法，为基因芯片制备和应用提供技术支持。方法利用交联聚合反应，在显微玻片表面衍生丙烯酸酯共聚物分子层，结合基因芯片技术制备寡核苷酸芯片。利用荧光标记的样品和CMT芯片杂交系统对所制备的芯片进行杂交实验，检测寡核苷酸在芯片表面的固定性能，并进行对照实验。结果杂交后的荧光检测表明寡核苷酸在丙烯酸酯共聚物表面上杂交点阵的荧光信号多，信号强度强，杂交点圆润、时一性好。结论利用化学交联法制备的芯片表面能用于寡核苷酸的固定且处理过程简单，为进一步运用到生物芯片制作打下了良好的基础。     

Microwave irradiation-accelerated in situ hybridization technique for HIV detection.

High frequency irradiation generated in a common household microwave oven was used to establish an in situ hybridization technique for rapid detection of HIV sequences in infected cells. A biotin-labeled DNA probe was subsequently detected either by an alkaline phosphatase-based colorimetric reaction or by fluorescence. When compared to standard hybridization procedures with radioactive or nonradioactive probes, microwave energy-mediated hybridization results in equal sensitivity and diminished background. The main advantage of this method, however, is the drastic reduction in time, allowing completion of the whole procedure, from sample preparation to hybrid signal visualization, within one hour. In addition to HIV detection, the approach described can be applied for the diagnosis of other viral infections and may stimulate the development of nucleic acid hybridization techniques based on microwave irradiation.     

人21号全染色体涂染探针的制备及其唐氏综合征诊断的应用研究

目的人21号染色体DNA涂染探针的制备及其应用于唐氏综合征诊断的研究。方法将显微分离的人21号染色体DNA进行简并寡核苷酸引物PCR（Degenerate Oligonucleotide Primed—PCR,DOP—PCR）扩增并标记制备成杂交探针后,与15例唐氏征疑似患者的外周血细胞核染色体进行荧光原位杂交（Fluorescence in situ hybridization,FISH）分析;同时以常规核型分析进行确诊并评估FISH结果。结果FISH分析诊断结果与常规核型分析一致,其中8例为非唐氏征患者,7例为唐氏征患者,准确率为100％,且非唐氏征患者与唐氏征患者细胞核中21号染色体的检出率分别高达99．12％和99．08％。结论制备的人21号全染色体DNA涂染探针能精确检测人类中期和间期细胞核中21号染色体的数目,该探针可用于唐氏综合征的诊断。     

Au nanoparticles enhanced fluorescence detection of DNA hybridization in picoliter microfluidic droplets

This work reports a facile microfluidic device for Au-nanoparticle enhanced fluorescence detection of tiny amount of nucleotides within droplets in a high-throughput way. Droplets containing single strand DNA probe and relevant complementary strands DNA(cDNA) are generated in flow-focusing manner and the hybridization between them is realized in droplets flowing along a long serpentine channel. In order to find the optimal experimental condition, finite element method simulation is used to predict the interface evolution between the two phase liquids. Based on the fluorescence emited by intercalator reacted with the generated double-strand DNA(dsDNA), the target cDNA with a concentration of 1nM can be detected in droplets. And when we adopt Au nanoparticles to immobilize DNA probe which can amplify the fluorescence intensity, 10pM completary DNA could be detected. Due to the advantages in high-throughput and compartmentalization of this droplet platform, the detection procedure can be finished in 3 h. Our method shows good potential application in facile, sensitive, low cost and fast DNA detection for applications in personal health care.     

The Application of Alkaline Phosphatase Labeled HBV Probe in Serum Detection

A method using non-radioactive material alkaline phosphatase to label HBV DNA as probe has been studied and used in clinical experiments to detect the HBV DNA in hepatitis serum. Alkaline phosphatase coupled with polyethyleneimine (PEI) using P-benzoquine as cross-linking reagent. The modified phosphatase was covalently linked to single strand DNA using glutraldehyde. Such single strand DNA enzyme complexes have been tested for blot hybridization, after hybridization and incubation with a substrate solution, sequences complementary to the probe can be visualized directly in 1 h. The minimum amount about 10 pg of target DNA has been detected in this way, 32P labeled probes are autoradiography 1 h after hybridization can only detect 10 ng, so the enzyme labeled probe is more sensitive than isotope labeled probe in 1 h fast test. Comparing the enzyme-labeled HBV DNA probe with 32P labeled the same one, positive proportion of detecting the HBV DNA in hepatitis patients was about 95.7%. Because the positive patient's serum detected by 32P labeled probe were selected through 1- week radiation, Alkaline Phosphatase labeled probes are color developed for only 1 h. Our experiment certified that it is a sensitive, specific, easy, rapid, safe and economical probe labeling and clinical virus DNA detection method.     

Cot-1 banding of human chromosomes using fluorescencein situ hybridization with Cy3 labeling

Summary We developed a new chromosome banding method byin situ hybridization of human Cot-1 DNA as a probe. Clear banding was produced on metaphase chromosomes of lymphoblastoid cells after probe detection with a fluorescent dye Cy3. Comparison with the known banding patterns revelaed a similarity to the R-banding with some significant differences: some centromeric heterochromatin regions show Cot-1 positive bands. This suggests that some repetitive sequences from the heterochromatin regions constitute a major component of Cot-1 DNA. This unique chromosome banding method, Cot-1 banding, may be used as a supplement to the conventional karyotype analysis. Scanning analysis of the fluorescence intensities of Cot-1 banding and Q-banding are useful for objectively analyzing the banding pattern including a detection of chromosome aberrations. The Cot-1 banding with Cy3 is particularly powerful when applied for the gene mapping by fluorescencein situ hybridization (FISH) because red fluorescence of Cy3 for chromosome staining can be readily distinguished from green fluorescence of fluorescein isothiocyanate (FITC) for probe labeling. Using this novel method, we mapped a 4 kb-DNA fragment from myelin protein zero (MPZ) gene on the chromosome 1q22 to q23.     

Technical Review: In Situ Hybridization

In situ hybridization is a technique that is used to detect nucleotide sequences in cells, tissue sections, and even whole tissue. This method is based on the complementary binding of a nucleotide probe to a specific target sequence of DNA or RNA. These probes can be labeled with either radio‐, fluorescent‐, or antigen‐labeled bases. Depending on the probe used, autoradiography, fluorescence microscopy, or immunohistochemistry, respectively, are used for visualization. In situ hybridization is extensively used in research, as well as clinical applications, especially for diagnostic purposes. This review discusses the basic technique of in situ hybridization. The standard in situ hybridization process is reviewed, and different types of in situ hybridization, their applications, and advantages and disadvantages are discussed. Anat Rec, 297:1349–1353, 2014. © 2014 Wiley Periodicals, Inc.     

Detection of varicella-zoster virus by dot-blot hybridization using a molecularly cloned viral DNA probe

Varicella-Zoster Virus (VZV) infection can be definitively diagnosed by isolation of virus in cell culture, a process that usually takes 7–14 days. In order to facilitate the more rapid detection of this virus, we developed a technique for hybridization of DNA from clinical specimens using an in vitro-labeled mixture of cloned fragments of VZV DNA as a probe. The assay can be completed in 36–48 hr and can be successfully carried out in the range of 10 pg to 10 ng of viral DNA. In analyses of 38 specimens from patients with a clinical diagnosis of VZV infection, the results of viral isolation and this assay were highly concordant. The sensitivity of standard cell culture for detection of VZV was 58%, whereas the sensitivity of the assay was 76%, not significantly different (P = 0.14). The specificity of cell culture was 100%, whereas that of the assay was 94% (P = 0.49). The technique appears to be sensitive, specific, and useful for analyses of tissues and body fluids.     

Role of multicolor fluorescence in situ hybridization (FISH) in simultaneous detection of probe sets for chromosome 18, X and Y in uncultured amniotic fluid cells.

Major aneuploidies diagnosed prenatally involve the autosomes 13, 18, and 21, and sex chromosomes. Fluorescence in situ hybridization (FISH) allows rapid analysis of chromosome copy number in interphase cells. The purpose of this study was to evaluate the role of multicolor fluorescence in situ hybridization in simultaneous detection of probe sets for chromosome 18, X, and Y in uncultured amniotic fluid cells as a safer alternative method for aneuploidy detection prenatally. Fifty amniotic fluid samples were analyzed by FISH and standard cytogenetics. Mean time to obtain results was three days for fluorescence in situ hybridization and 20 days for karyotype. Fluorescence in situ hybridization was informative in 43 samples (86%), and within this group, two aneuploidies were correctly identified. This evaluation demonstrates that FISH with X, Y, and 18 alpha satellite DNA probes could accurately and rapidly detect aneuploidies involving these chromosomes and could be used in any prenatal clinical laboratory.     

Chemiluminescent microwell hybridization assay for direct detection of human parvovirus B19 DNA

A method for the direct detection of human parvovirus DNA in serum samples that uses a digoxigenin-labeled RNA probe to hybridize with target B19 DNA, followed by capture of the hybrid onto a microtiter plate wells previously coated with a second oligonucleotide probe was developed. The captured hybrid is then detected with anti-digoxigenin-alkaline phosphatase conjugate and Chemiluminescent substrate and the reaction read on a scintillation counter. The relative sensitivities of the microwell and standard dot blot hybridization assays were compared. The chemiluminescent microwell hybridization assay was more sensitive than dot-blot hybridization and could be performed in a few hours. This format, therefore, permits rapid and sensitive detection of parvovirus DNA suitable for the clinical setting.     

Rapid antiviral DNA-DNA hybridization assay for human cytomegalovirus

A rapid DNA-DNA hybridization technique that can be accomplished in 4 to 5 days was compared with plaque reduction assay to determine its reliability in performing antiviral assays for human cytomegalovirus (HCMV). The assay involves lysing infected cells, direct wicking of denatured DNA onto membranes and hybridization using a 125I-labeled HCMV DNA probe. Using ten ganciclovir sensitive clinical HCMV strains for comparison, the DNA hybridization technique correlated well with the plaque assay. Clinical HCMV strains previously identified as resistant to ganciclovir were also readily identified. The DNA-DNA hybridization assay is less tedious and more rapid than plaque reduction assays, and thus, provides an excellent alternative for evaluation of the antiviral activity of drugs against HCMV.