【作者】 习东；

【导师】 宁琴；

【作者基本信息】 华中科技大学 ， 免疫学， 2007， 博士

【摘要】 【背景及目的】乙型、丙型病毒性肝炎是分别由乙型肝炎病毒（hepatitis B virus, HBV）、丙型肝炎病毒（hepatitis C virus, HCV）引起的危害严重的传染病。我国是乙型肝炎和丙型肝炎高发区。乙型肝炎和丙型肝炎的合并感染率高,其中尤以药瘾者及输血者的合并感染率为最高。目前,检测HBV脱氧核糖核酸（deoxynucleic acid, DNA）、HCV核糖核酸（nucleic acid, RNA）是判断病毒复制水平,观察疗效和评估预后的主要指标。病毒核酸的检测方法主要有两类,一类是聚合酶链反应（PCR）,另一类是核酸杂交。临床检验的常规方法是PCR,PCR对实验室环境要求高,结果可出现假阳性,作为临床常规检验手段尚有争议。目前常规检测HBV和HCV的PCR技术是用不同的反应体系分别扩增血清标本中病毒核酸,既繁琐又增加污染机会。为实现同步检测HBV和HCV,研究人员用多重PCR在一个反应体系内同时扩增HBV和HCV的病毒核酸,已取得了一定成效,但用多重引物对多个模板同时扩增具有较大难度,特别是HCV感染者血清中病毒浓度较低。核酸杂交需要检测核酸的基因探针,如放射性标记基团、荧光基团、化学发光基团。目前,放射性标记探针已被非放射性标记取代,但荧光基团、化学发光基团易于淬灭,应用具有局限性。因此有必要研发特异、敏感、经济、安全并方便于临床使用的检测探针和技术。DNA分子的大小在纳米尺度内。近年来兴起的DNA纳米技术是将DNA结合到纳米颗粒上构成纳米颗粒基因探针,通过与靶DNA之间的互补实现纳米颗粒的组装,已成为一种新型的DNA检测系统。具有高电子密度的金（Au）纳米颗粒在透射电镜下易于观察,Au纳米颗粒聚集为微米大小即呈现肉眼可见的红色,银染色可放大检测信号。除Au之外,多种纳米材料已被开发,其中核/壳结构的纳米颗粒备受瞩目。本课题通过化学方法制备Au、Fe₃O₄（核）/Au（壳）纳米颗粒HBV DNA、HCV cDNA基因探针,通过尼龙膜上斑点杂交或透射电镜观察液相中杂交聚集体的方法检测病毒核酸,以期建立纳米颗粒直接检测病毒基因的方法。同时,利用Fe₃O₄（核）/Au（壳）纳米颗粒基因探针在磁场中的特殊性质,通过高梯度磁场进行杂交聚集体的分离,探索Fe₃O₄（核）/Au（壳）纳米颗粒基因探针通过磁场检测病毒基因的新方法。进而,我们以检测HBV、HCV双重感染为目的,对纳米金比色芯片同步目视化检测方法的可行性和有效性进行研究,并探讨用Au纳米颗粒基因探针在透射电镜下同步检测HBV、HCV双重感染的方法。【方法】分别用柠檬酸还原金氯酸法、化学共沉淀法制备Au、Fe₃O₄纳米颗粒,二法结合制备Fe₃O₄（核）/Au（壳）纳米颗粒。通过金-硫（Au-S）共价键,将烷氢硫醇修饰的寡核苷酸结合在Au、Fe₃O₄（核）/Au（壳）纳米颗粒上,制备Au、Fe₃O₄（核）/Au（壳）纳米颗粒HBV DNA、HCV cDNA基因探针,紫外-可见分光光度计进行波长扫描。用荧光标记法检测纳米颗粒基因探针表面寡核苷酸的覆盖数和与其互补的寡核苷酸的杂交效率。提取乙型肝炎病人血清中HBV DNA,PCR扩增。同时提取乙型、丙型肝炎合并感染病人血清中的病毒DNA和RNA,利用多重PCR方法扩增HBV DNA和HCV cDNA。纳米颗粒基因探针检测病毒核酸的方法有三种:方法一为检测探针与固定在尼龙膜上的捕捉链构成双探针,用斑点杂交法检测HBV DNA的PCR产物或者HBV DNA和HCV cDNA的多重PCR产物,加入银离子（Ag+）-对苯二酚液染色观察结果;方法二为在含有Au或者Fe₃O₄（核）/Au（壳）纳米颗粒基因探针的液相检测体系中加入HBV DNA和/或HCV cDNA,透射电镜下观察;方法三是将Fe₃O₄（核）/Au（壳）纳米颗粒基因探针杂交体系通过高梯度磁场,检测磁场处理前后Au特征性吸收峰值的改变。【结果】制备的Au纳米颗粒、Fe₃O₄（核）/Au（壳）纳米颗粒粒径分别为（10±5）nm、（15±5）nm,水相分散良好。Au、Fe₃O₄（核）/Au（壳）纳米颗粒探针表面寡核苷酸的最大覆盖数分别为（132±10）条、（120±8）条,最大杂交效率分别为（22±3）%、（14±2）%。Au、Fe₃O₄（核）/Au（壳）纳米颗粒分别在520nm、522 nm有最大吸收峰,经寡核苷酸修饰后,最大吸收峰分别改变为524nm、525 nm。Au、Fe₃O₄（核）/Au（壳）纳米颗粒在尼龙膜上用斑点杂交法可分别检出低至10fmol、100 fmol的合成靶DNA,可目视化检出乙型肝炎患者血清中HBV DNA的PCR产物。在纳米颗粒基因探针液相检测体系中加入HBV DNA,透射电镜观察到纳米颗粒组装成大的网状结构的聚集体。通过高梯度磁场可以将磁性探针与靶DNA形成的杂交聚集体与对照进行区分。同时提取获得乙型、丙型肝炎合并感染病人血清中病毒RNA及DNA,多重PCR同时扩增HBV DNA和HCV cDNA,纳米金比色芯片可以同步目视化检出乙、丙型肝炎合并感染病人血清中HBV DNA和HCV RNA的PCR产物。使用Au纳米颗粒基因探针通过透射电镜可同步检出乙、丙型肝炎合并感染患者血清中提取的HBV DNA和HCV RNA。【结论】1、成功制备了Au、Fe₃O₄（核）/Au（壳）纳米颗粒基因探针,它们可直接用于肝炎病毒的检测。2、通过芯片分析技术可实现目视化检测HBV DNA的PCR产物或乙、丙型肝炎合并感染患者的HBV DNA和HCV RNA的多重PCR产物,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,可望在许多领域、尤其是病毒基因检测领域有广泛用途,此类基因芯片在多基因检测芯片上有潜在的应用价值。3、通过透射电镜,使用纳米颗粒基因探针可直接检测乙肝病人阳性血清中提取的HBV DNA,可同步检出乙、丙型肝炎合并感染患者血清中提取的HBV DNA和HCV cDNA,甚至可达到无需PCR水平,此方法具有敏感性高、特异性好的特点。4、Fe₃O₄（核）/Au（壳）纳米颗粒基因探针杂交体系通过高梯度磁场可以将磁性探针与靶DNA形成的杂交聚集体与对照进行区分,表明磁性纳米颗粒基因探针结合磁场应用进行核酸检测简单、易行、实用。更多还原

【Abstract】 Sequence-specific methods for detecting polynucleotides are critical to the diagnosis of genetic and pathogenic diseases. Most detection systems make use of the hybridization of a target polynucleotide with oligo- or polynucleotide probes containing covalently linked reporter groups, which include radioactive labels, fluorescent labels and chemilumine- scence schemes, etc. Each of these strategies has advantages and disadvantages and no single method has gained supremacy. During the last decade, there has been an increasing interest in using nanoparticles in DNA detection. In the present paper, we prepared gold or gold-coated iron oxide nanoparticle Hepatitis B virus （HBV） DNA probes and using these probes we established a methology to detect HBV DNA molecule extracted from hepatitis B patient on nylon membrane by blot-hybridization or in liquid media by TEM. We have examined the feasibility of the detection of HBV DNA with gold-coated iron oxide nanoparticles and magnetic separator. And we further established a simple and rapid visual method based on either DNA microarray or TEM with nanogold supported probes for detecting HBV and HCV simultaneously.Au nanoparticles were produced via citrate reduction of tetrachloroauric acid （HAuCl4）. Superparamagnetic iron oxide nanoparticles （SPION） were produced with chemical precipitation. The coating of Au on magnetic nanoparticles was performed via citrate reduction of HAuCl4 on the surface of SPION that served as seeds. Alkanethiol modified oligonucleotide was bound with self-made Au and Fe₃O₄（core）/Au（shell）nanoparticles to form nanoparticle HBV DNA gene probes through covanlt binding of Au-S. With a fluorescence-based method, for Au nanoparticles and Fe₃O₄（core）/Au（shell） nanoparticles, the maximal surface coverage of hexaethiol 30-mer oligonucleotides was （132±10） and （120±8） oligonucleotides per nanoparticle, and the maximal percentage of hybridization strands on nanoparticles was （22±3）% and （14±2）%, respectively. Based on a two-probe sandwich hybridization/ nanoparticle amplification/ silver staining enhancement method, Au nanoparticle and Fe₃O₄（core）/Au（shell） nanoparticle gene probes could detect as low as 10-11mol/L and 10-10mol/L composite HBV DNA molecules on nylon membrane, respectively, and the PCR products of HBV DNA visually. As evidenced by transmission electron microscopy, the nanoparticles assembled into large network aggregates when nanoparticle HBV DNA gene probes were applied to detect HBV DNA molecules.HBV DNA and HCV RNA extracted from serum in patients with HBV and HCV coinfection can be specifically amplified simultaneously in one system by multi-primer PCR. With the aid of Au nanoparticle-supported mercapto-modified oligonucleotide serving as detection probe, and oligonucleotide immobilized on nylon membrane surface acting as capturing probe, HBV and HCV PCR products were detected visually by sandwich hybridization based on highly sensitive aggregation and silver staining. As evidenced by transmission electron microscopy, large network aggregates assembled by nanoparticles identifying HBV and HCV simultaneously in serum in patients with HBV and HCV coinfection were seen clearly.Our results suggested that DNA modified nanoparticle probe can be used to detect virus nucleic acid. The established oligonucleotide array for the detection of HBV and HCV coinfection is convenient and efficient with high specificity. The detection- visuallized method has many advantages, including high sensitivity, simple operation and low cost. This technique has potential applications in many fields, especially in multi-gene detection chips. It provides a nationale for the measurement of other viruses coinfection. Transmission electron microscopy can detect HBV or HBV and HCV simultaneously in serum in patients. Detecting DNA with iron oxide nanoparticles and magnetic separator was feasible and might be an alternative effective method.更多还原