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类型 基础研究 预答辩日期 2018-03-09
开始(开题)日期 2011-05-27 论文结束日期 2017-08-25
地点 逸夫科技馆三楼会议室 论文选题来源 973、863项目     论文字数 9 (万字)
题目 基于纳米磁分离和化学发光的肝炎分子检测新技术研究
主题词 磁性纳米颗粒,化学发光,肝炎,分子检测
摘要 随着纳米技术的迅速发展,纳米材料逐渐被应用到生命科学领域,为其研究和发展提供了新的技术和手段。磁性纳米颗粒(magnetic nanoparticles, MNPs)作为纳米材料的一个重要组成部分,目前已被广泛应用于各种生物分子的检测等方面。化学发光具有安全、灵敏度高,线性范围宽等特点,为病毒检测提供了一个良好的平台。本论文以肝炎为检测对象,结合化学发光和磁分离技术的优点,建立了几种快速、高通量、灵敏和实用的肝炎分子检测新技术。主要包括以下七部分内容: 1. 功能化磁性纳米颗粒的制备及在核酸提取中应用 本课题在进行乙肝样本高灵敏检测研究前对功能化磁性纳米颗粒的制备进行了改进,采用软模板法制备出的MNPs具有明显的核壳结构。将磁性纳米颗粒应用于细菌和全血样本核酸提取中均获得良好的提取效果,有望开发出磁珠分离法核酸提取试剂盒。 2. 基于功能化磁性纳米颗粒的肝炎核酸分子提取及扩增 针对不同来源的两种乙肝核酸提取方法提取效果进行比较分析,并对提取出来的乙肝核酸样品进行验证性实验分析。3. 基于纳米磁分离和化学发光的乙肝PCR扩增检测方法的建立及优化 本章以人工合成的生物素标记的乙肝模拟核酸片段ssHBV DNA为目标分子,建立了一种乙肝核酸分子的化学发光杂交检测方法,结果表明,该方法的特异性较好。通过对检测体系中涉及到的多种实验条件进行优化处理,对整个检测方法有了更深入的了解,并得出了最优化的实验条件,有望提高该方法的检测灵敏度。 4. 基于纳米磁分离和化学发光的丙肝RT-PCR扩增检测方法的建立 基于前期对乙肝病毒的分子检测技术,我们建立一种基于磁分离和化学发光信号放大并适用于检测RNA病毒核酸检测方法。我们以HCV RNA为研究对象,以丙肝RNA特征基因为检测对象,在磁珠法提取病毒核酸DNA后进行RT-PCR扩增标记获得靶序列,通过两者杂交后进行化学发光检测特异性。 5. 基于纳米磁分离和化学发光的乙肝全基因扩增检测方法的建立及优化 以磁珠法全血乙肝提取DNA做模板,同时以GADPH基因探针为内标,进行生物素标记的包含乙肝HBV的全血全基因组扩增,从而建立了一种乙肝核酸全基因扩增标记的化学发光杂交检测方法。通过全基因组扩增技术进行乙肝HBV的化学发光检测,有利于进一步提高该方法的检测灵敏度。 6. 基于环介导等温扩增和化学发光的病毒检测方法初探 探索了一种基于逆转录环介导等温扩增(RT-LAMP)和化学发光相结合的办法检测RNA病毒。优化了HBV和HCV的LAMP扩增和探针杂交的相关条件,如LAMP反应温度、杂交时间、杂交温度、探针浓度等。本方法相比于传统的基于PCR的化学发光检测,检测时间缩短了近1 h。此方法因基于LAMP扩增和核酸杂交,故在核酸检测方面具有高度特异性。此方法简化了检测过程,更容易实现自动化,因此在临床上有着广泛的应用前景。 7. 功能化微球在乙型肝炎病毒表面抗原的分子检测应用探索研究 制备了一种新型的、无皂乳液聚合技术,制备了粒径合理(约400 nm)的单分散氨基官能化聚合物微球。通过扫描电子显微镜(SEM)、Zeta电位和傅立叶变换红外光谱仪(FT-IR)等各种表征方法表明,氨基基团已成功地引入到聚苯乙烯的微球表面,而且所制备的氨基化微球具有均匀的尺寸和良好的分散性等特性。随后通过将这种功能化微球固定化单克隆抗体并富集成功,从而以制备的功能化微球作为载体建立了一种基于化学发光酶联免疫分析法(ELISA)方法应用于乙肝病毒表面抗原(HBsAg)的分子检测新技术。这种新的化学发光ELISA检测证明具有较好的特异性,且在使用ALP-AMPPD特定的化学发光系统中HBsAg的分子检测灵敏度较高。
英文题目 Novel Methods for Molecule Detection of Hepatitis Viruses Based on Magnetic Nanoparticles Separation and Chemiluminescence
英文主题词 Magnetic nanoparticles; Chemiluminescence; Hepatitis; Molecular detection
英文摘要 With the rapid development of nanotechnology, nano materials have been applied to the field of life science. Magnetic nanoparticles (magnetic, MNPs), as an important component of nanomaterials, have been widely used in the detection of various biological molecules, such as nanoparticles. Chemiluminescence has the advantages of high security, high sensitivity and wide linear range, which provides a good platform for virus detection. In this paper, we have established several rapid, high-throughput, sensitive and practical techniques for the detection of hepatitis by combining the advantages of chemiluminescence and magnetic separation. Specific contents include: 1. Preparation of functionalized magnetic nanoparticles and their application in nucleic acid extraction In order to improve the magnetic nanoparticles functionalized with probe, in this paper the high sensitive detection of hepatitis B virus samples of functional magnetic nanoparticles before preparation was improved by MNPs prepared by soft template method has obvious core-shell structure, the average diameter is 500 nm, and the circular particles, uniform size, superparamagnetism, saturation magnetization of 1.7374 emu/g, prepared by SiO2 coated Fe3O4 composite particles, the average particle size is 700 nm, with good dispersion, particle size uniformity is round. The magnetic nanoparticles were applied to the extraction of nucleic acids from bacteria and whole blood samples, and good extraction results were obtained. 2. Extraction and amplification of nucleic acid molecules based on functionalized magnetic nanoparticles Firstly, two kinds of hepatitis B nucleic acid extraction methods were compared and analyzed, and the results were analyzed. The results showed that although the amount of nucleic acid extracted from serum hepatitis virus was small, it could be used in PCR amplification, but it was less effective in whole genome amplification. At the same time, the amount of nucleic acid extracted from the whole blood is high, on the one hand, it can be used for PCR amplification, and can also be applied to the whole genome amplification technology. Whole genome amplification of whole blood hepatitis B nucleic acid DNA can also be used for PCR amplification. In this chapter, we also optimized the whole genome amplification condition of whole blood hepatitis B virus DNA, which laid the foundation for the high-throughput detection of hepatitis B virus detection by chemiluminescence. 3. Establishment and optimization of PCR amplification detection method based on magnetic separation and chemiluminescence This chapter takes the synthetic biotin labeled hepatitis B nucleic acid fragment ssHBV simulation DNA as the target molecular, chemical luminescence hybridization detection method, a hepatitis B nucleic acid molecules were established. The results show that the good specificity of the method. To optimize the processing through a variety of experimental conditions on the detection system involved in, have a deeper understanding on the detection method, and obtains the optimized experimental conditions, is expected to improve the detection sensitivity of this method. The following optimized conditions: the best particle content is 100 g; the optimal concentration of SA MNPs was 0.1 mM; the optimal concentration of amino modified probe into 2 M; the best hybridization temperature of 45 C; the best time for hybridization was 30 min; the method in the low concentration range of 10-10000 copies/mL, a linear relationship the signal intensity, the sensitivity was 10 copies/mL. 4. Detection of hepatitis C virus RT-PCR amplification based on magnetic separation and chemiluminescence Hepatitis C is a kind of hepatitis caused by RNA virus, which is different from hepatitis B through the HBV DNA virus replication, its infectivity and concealment is strong, social harm is bigger. Based on the early detection of hepatitis B virus, we set up a method for detecting RNA virus nucleic acid based on magnetic separation and chemiluminescence signal amplification. We use HCV RNA as the research object, with the C RNA feature gene for object detection, extraction of virus nucleic acid in DNA magnetic beads method for RT-PCR amplification. The target sequence, through both hybridization after specific chemiluminescence detection. 5. Establishment and optimization of the detection method for hepatitis B virus gene amplification based on magnetic separation and chemiluminescence The whole blood by magnetic beads method to extract DNA template of hepatitis B, and GADPH gene probe as an internal standard, were labeled with biotin containing hepatitis B HBV blood, whole genome amplification, so as to establish a chemical marker of hepatitis B nucleic acid gene amplification luminescent hybrid detection method. The detection of hepatitis B virus HBV by whole genome amplification technology is helpful to improve the detection sensitivity. The results showed that the specificity of the method was good, the hybridization efficiency of chemiluminescence is greatly improved, to optimize the experimental conditions and results are as follows: the optimum particle content is 80 g; the optimal concentration of SA MNPs was 0.1 mM; the optimal concentration of amino modified probe into 100 M; the optimal hybridization time was 40 min. 6. Method of virus detection based on loop mediated isothermal amplification and chemiluminescence The loop mediated isothermal amplification (LAMP) technique allows nucleic acids to be amplified in a fast and highly specific manner under isothermal conditions, avoiding the long and complex thermal cycling of PCR and expensive experimental instruments. In this paper, a method based on reverse transcription loop mediated isothermal amplification (RT-LAMP) and chemiluminescence was used to detect RNA virus. Biotin-11-dUTP joined in LAMP amplification to PCR products with biotin labeled, after hybridization with specific probes, finally using ALP- AMPPD chemiluminescence reaction system to determine if the samples contain viral nucleic acid, in order to achieve the purpose of pathogen detection. In this study, we optimized the conditions of LAMP amplification and probe hybridization, such as LAMP reaction temperature, hybridization time, hybridization temperature, probe concentration and so on. The reaction temperature is 63 C on HA and NA gene amplification efficiency; probe concentration is 10 M, the hybridization time was 50 min for HA and NA probe the best effect; however, two gene probes the optimal hybridization temperature is slightly different, the optimum temperature of 50 hybrid HBV genes, the optimum temperature is 55 hybridization probe for HCV. Finally, through the sensitivity test, we obtain that this method can detect 103 copies / mL of HCV-RNA. Compared with the traditional PCR based chemiluminescence detection, the detection time was shortened by nearly 1 h. This method is highly specific for nucleic acid detection based on LAMP amplification and nucleic acid hybridization. This method simplifies the detection process and makes it easier to automate, so it has a broad application prospect in clinic. 7. Application of Functional Microsphere in Human Hepatitis B Virus Surface Antigen Detection A novel and simple emulsifier-free emulsion polymerization technique was developed for preparation of mono-dispersed amino functionalized polymer microspheres with well defined diameters (about 400 nm). Various characterization methods demonstrated that the obtained amino microspheres had a uniform size and good dispersity which were confirmed by scanning electron microscope (SEM). Zeta potential and Fourier transform infrared spectrometer (FT-IR) demonstrated that amino groups have been successfully introduced to the microsphere surface. These functionalized microspheres have been shown to be efficient and controllable carriers capable of immobilizing and enriching monoclonal antibodies. Moreover, a newest chemiluminescent enzyme-linked immunoassay (ELISA) approach has been developed for human Hepatitis B virus surface antigen (HBsAg) detection. HBsAg was sandwiched between goat anti-HBsAg polyclonal antibody coated on microspheres and mouse anti-HBsAg antibody. Alkaline phosphatase (ALP) conjugated horse anti-mouse immunnogloblin was used to bond with monoclonal antibody. Finally, chemiluminesent (CL) signals were recorded after adding 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy) phenyl-1,2- dioxetane (AMPPD) which was used as a chemiluminescent substrate reagent of ALP. This novel chemiluminescent ELISA assay was proved to be of excellent specificity and high sensitivity when using ALP and AMPPD luminescence systems for specific HBsAg detection.
学术讨论
主办单位时间地点报告人报告主题
东南大学和华中科技大学 2009.11.12 东南大学榴园新华厅 孙啸 教授 基因转录调控的生物信息分析
生物电子学国家重点实验室 2010.3.25 东南大学春晖堂 孙清江 教授 生物/纳米材料/太阳能电池
东南大学生物电子学国家重点实验室 2010.3.31 生物电子国家重点实验室三楼会议室 张晓晖 老师 蛋白质结构与功能的单分子研究
东南大学生物科学与医学工程学院何农跃课题组 2010.4.1 生物电子学实验室3楼会议室 马超 基于磁性纳米颗粒的乙肝病毒化学发光检测
东南大学生物科学与医学工程学院何农跃课题组 2010.5.28 生物电子学实验室3楼会议室 马超 磁珠法全血DNA提取试剂盒的研发及应用推广
上海交通大学微纳研究院 2010.8.6 上海交通大学微纳研究院会议室 马超 磁性粒子基础上高灵敏乙肝核酸检测方法与试剂的研制
东南大学生物电子学国家重点实验室 2011.10.22. 生物电子学实验室3楼会议室 马超 Preparation of Magnetic Nanoparticles and Its Applications in Molecular Detection of Pathogen and Virus Based on Chemiluminescence and WGA Methods
东南大学生物科学与医学工程学院 2012.2.28 丁家桥校区综合楼307教室 Hanry Yu教授 Mechanobiology: an integrative and quantitative approach to understand biological functions and translation into biomedical applications
东南大学生物科学与医学工程学院 2011.4.15 生物电子学国家实验室三楼会议室 张岩 博士 纳米光子学在生物医学中的应用
东南大学生物电子学国家重点实验室 2010.9.15 生物电子学实验室3楼会议室 郑杰 教授 Detecting sequence polymorphisms associated with recombination hotspots in human genome
     
学术会议
会议名称时间地点本人报告本人报告题目
第七届国际后基因组生命科学技术学术论坛 2011.10.28 西南大学 Comparison and verification of Genomic DNA extraction based on magnetic nanoparticles from Escherichia coli and whole blood
第241届美国化学学会年会 2011.3.25 阿纳海姆(美国) Genomic DNA extraction and purification based on magnetic nanoparticles from Human Being’s whole blood
第243届美国化学学会年会 2012.3.25 奥兰多(美国) Chemiluminescene detection of HBV gene based on magnetic nanoparticles
2010’全国纳米生物与医学学术会议 2010.10.22 武汉大学 Magnetic nanoparticle-based genomic DNA extraction and purification from the whole blood of the Human Being
第六届国际后基因组生命科学技术学术论坛 2009.9.18 清华大学(北京西郊宾馆) Comparison of three whole genome amplification (WGA) methods of genomic DNA from Escherichia coli
2011中国生物医学工程学术年会会议 2011.11.2 华中科技大学 基于磁性纳米颗粒的核酸提取及扩增检测技术
2011年江苏省生物工程年会 2011.11.26 东南大学榴园宾馆 磁性纳米颗粒的核酸提取、酶切及扩增检测技术研究
中国-新家坡生物医学工程国际交流会 2011.12.3 东南大学榴园宾馆 Comparison and Verification of Genomic DNA Extraction Based on Magnetic Nanoparticles from Bacteria and Whole Blood
     
代表作
论文名称
Preparation and Characterization of Monodisperse Core–Shell Fe3O4@SiO2 Microspheres and Its
Magnetic Nanoparticles-Based Extraction and Verification of Nucleic Acids from Different Sources
Chemiluminescence Detection of Whole Blood Based on Magnetic Nanoparticles and Whole Genome Amplific
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
孙啸 正高 教授 博导 东南大学
肖鹏峰 正高 教授 博导 东南大学
史智扬 正高 主任医师 博导 江苏省疾病预防控制中心
王念跃 正高 主任医师 博导 南京市第二医院
王志飞 正高 教授 博导 东南大学
      
答辩秘书信息
姓名职称工作单位备注
王遵亮 副高 副教授 东南大学