基于磁性纳米颗粒和化学发光的多种病毒同时检测方法的研究

发布时间：2018-07-03 20:14

本文选题：基于 + 磁性　；参考：《东南大学》2015年博士论文

【摘要】：基于磁性纳米颗粒的细胞,细菌和病毒核酸的提取方法研究核酸(NA)提取是进行遗传研究和DNA传感器开发的关键步骤。核酸抑制剂的存在会妨碍核酸的下游应用。这些盐离子和其他有机污染物给DNA生物传感器的开发及灵敏的分析提出了挑战。我们提出了基于磁珠分离核酸的方法,并对可能影响核酸分离的因素进行了优化。结果表明,6M的胍盐酸盐的浓度为分离核酸的最优浓度,核酸的裂解缓冲液和浓度和pH与核酸产量成负相关,核酸产量为在pH 4-5的环境下相对稳定。可以看出,使用carrier RNA在病毒基因组分离中是十分重要的。当加入1：1的比例乙醇至溶解物能够大大提高核酸产量。无DNA酶和RNA酶水,1×TE缓冲液和2×PCR缓冲液洗脱效率进行比较可以看出,carrier RNA在DNA酶和RNA酶自由水和1×TE缓冲液中洗脱效率最佳。从大量的细胞中提取核酸用较大的洗脱缓冲液的体积洗脱效果更佳。本研究建立了一种简单的方法,从细胞,细菌和血清提取核酸。将纯化的核酸成功扩增PCR中和RT-PCR,以验证此方法的可靠性。使用纳米颗粒可以促进使用这种方法在自动化核酸提取仪开发和应用,可用于高通量半自动或全自动诊断系统的发展。基于PCR扩增和化学发光乙肝病毒的高灵敏度检测HBV感染已被认为可以引发一系列严重的疾病,如肝细胞癌(HCC),肝内胆管细胞癌和非霍奇金淋巴瘤。对这一感染的早期诊断对疾病的治疗以及抑制疾病传播至关重要。目前的分子检测系统价格昂贵,而且需要高技能的劳动力进行测试和处理数据。本研究提出了一个通过结合磁分离,多重PCR和化学发光技术的高灵敏度、可自动化检测临床样品乙肝病毒检测方法。对可能影响PCR的因素,如引物浓度,PCR缓冲液浓度,氯化镁浓度(MgCl2),引物的Taq DNA聚合酶的浓度和PCR的退火温度进行优化,以获得最大的产量。探针-模板杂交温度进行了探索研究,最终得到杂交温度为45℃。优化的探针浓度为5μM。最佳杂交时间被确定为30分钟。用50μg探针修饰的磁珠产生最大化学发光信号。当用HCV和HIV感染的血清替换HBV血清时,检测获得阴性信号。该方法可检测10病毒拷贝/毫升血清的HBV病毒,有很好的灵敏度。基于PCR扩增和化学发光丙肝病毒的高灵敏度检测丙型肝炎病毒(HCV)是全世界的主要威胁之一。感染了这种病毒会导致慢性肝炎,肝硬化和原发性肝细胞癌(HCC)的发生。研发成本低廉的诊断工具,以便及早发现丙型肝炎病毒的发展是在发展中国家是非常必要的。本研究建立了一种基于磁性纳米颗粒和化学发光的HCV检测方法。利用一步法逆转录聚合酶链反应(RT-PCR)对HCV-RNA进行扩增,以实现较高的灵敏度的检测。PCR的引物退火温度被确定为58℃。在1.5×PCR缓冲液浓度下,获得最佳的扩增效果。同时对MgCl2和引物的浓度进行了优化,最佳浓度分别2.4州和200 nM。热启动Taq DNA聚合酶的最佳用量为1.5U/50μl。探针和模板杂交的最佳在温度为55℃。最适探针浓度为5μM。当杂交在30分钟时记录得到最大的化学发光信号。该方法成功地检测到10病毒颗粒/毫升血清,有较好的特异性和灵敏度。基于磁性分离和化学发光的艾滋病毒检测方法研究人类免疫缺陷病毒(HIV)是一种感染人类免疫系统细胞的致病病毒。自从艾滋病病毒被发现至信今已造成超过2500万人死亡,每年报告新增艾滋病毒感染者270万。因此,研究一种新的廉价有效的HIV病毒诊断方法极为重要。本研究介绍了一种基于DNA杂交和化学发光的HIV检测分析。利用一步法反转录聚合酶链反应(RT-PCR)用于扩增的HIV靶序列。最佳退火温度为58℃。Biotin-11-dUTP掺入到一步法RT-PCR扩增反应中,使扩增产物标记上Biotin。探针修饰的磁珠被用来捕获这些靶序列,并生成检测信号。本论文对可能会影响化学发光信号的不同因素进行了优化,最后得到最佳杂交温度是35℃。5μM的探针浓度产生最大的化学发光信号。最佳杂交时间为30分钟。这种方法显示出良好的特异性。当HCV和HBV感染的血清替换HIV血清时,得到的阴性信号。该系统成功地检测到10病毒拷贝／毫升HIV血清。基于磁性纳米颗粒和化学发光分析同时提取和检测多种病毒相比于酶联免疫(ELISA)测定方法,基于核酸检测的方法更为灵敏。多种试验方法已经被用于检测多种病原体,并能降低核酸检测的成本。但是,这些测定法无法可靠地检测大量病原体。在本研究中,建立了一个基于DNA杂交的化学发光检测方法,被并用于同时检测多种病原体而对病原体的数目没有限制。通过开展HBV, HCV和HIV的同时提取和检测,探头具有较高的特异性可捕获病毒序列。在其它病毒有更高负载的存在时仍可检测到单个病毒,该试验可以检测10拷贝/毫升血清10拷贝／毫升HCV血清和100拷贝/毫升HIV血清。基于磁分离技术从核酸提取到化学发光检测,该方法可以整合到一个自动化系统用于便捷地高通量病原休检测。
[Abstract]:Extraction of cells, bacteria and virus nucleic acids based on magnetic nanoparticles study of nucleic acid (NA) extraction is a key step in genetic research and the development of DNA sensors. The presence of nucleic acid inhibitors hinders the downstream application of nucleic acids. The development and sensitive analysis of these salt ions and other organic pollutants to DNA biosensors The challenge. We propose a method based on magnetic bead separation of nucleic acid and optimize the factors that may affect nucleic acid separation. The results show that the concentration of guanidine hydrochloride in 6M is the optimal concentration of nucleic acid separation, the buffer solution and the concentration and pH of nucleic acid are negatively correlated with the yield of nucleic acid, and the yield of nucleic acid is relatively stable in the environment of pH 4-5. It is evident that the use of carrier RNA is very important in the isolation of the virus genome. When the proportion of ethanol into the 1:1 can greatly increase the yield of nucleic acid, no DNA and RNA water, 1 x TE buffer and 2 x PCR buffer elution efficiency can be compared, carrier RNA is found in the DNA enzyme and RNA enzyme free water and 1 * TE buffer solution. Elution efficiency is best. Extraction of nucleic acids from a large number of cells is better than the size of a larger elution buffer. A simple method is established to extract nucleic acids from cells, bacteria and serum. The purified nucleic acid is successfully amplified by PCR neutralization and RT-PCR to verify the reliability of the method. This method is developed and applied in an automated nucleic acid extraction instrument, which can be used for the development of high throughput semi-automatic or fully automated diagnostic systems. Detection of HBV infection based on PCR amplification and high sensitivity of chemiluminescent HBV has been considered to lead to a series of serious diseases, such as hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma and non Hodgkin drenching. The early diagnosis of this infection is essential for the treatment of the disease and the suppression of the spread of the disease. The current molecular detection system is expensive and requires highly skilled labor to test and process data. This study proposes a high sensitivity by combining magnetic separation, multiple PCR and chemiluminescence technology, which can be automated. Detection of hepatitis B virus detection methods in clinical samples. The factors that may affect PCR, such as primer concentration, PCR buffer concentration, magnesium chloride concentration (MgCl2), Taq DNA polymerase and PCR annealing temperature, are optimized to obtain the maximum yield. Probe template hybridization temperature was explored, and finally the hybridization temperature was 45 The optimum hybridization time of the probe was 5 mu M. and the best hybridization time was determined to be 30 minutes. The maximum chemiluminescence signal was produced by the magnetic beads modified with 50 micron g probes. When the serum HCV and HIV infected serum were replaced with HBV sera, the negative signals were detected. This method can detect the HBV virus of the copy / milliliter serum of the virus. It has a good sensitivity. Based on the PCR expansion, the method has a good sensitivity. The high sensitivity of increasing and chemiluminescence hepatitis C virus (HCV) detection is one of the major threats to the world. Infection of the virus can lead to chronic hepatitis, cirrhosis and primary hepatocellular carcinoma (HCC), and the development of low cost diagnostic tools for the development of hepatitis C virus in developing countries is in developing countries. It is necessary to establish a HCV detection method based on magnetic nanoparticles and chemiluminescence. HCV-RNA is amplified by one step reverse transcriptase polymerase chain reaction (RT-PCR) to achieve high sensitivity. The annealing temperature of the primer of.PCR is determined to be 58. The optimum results are obtained at the concentration of 1.5 x PCR buffer. At the same time, the concentration of MgCl2 and primers was optimized. The optimum concentration of the optimum concentration of 2.4 state and 200 nM. Taq DNA polymerase was the optimum at the 1.5U/50 Mu L. probe and the template hybridization at the temperature of 55. The optimum probe concentration was 5 mu M. when the maximum chemiluminescence signal was recorded when the hybridization was 30 minutes. The human immunodeficiency virus (HIV) is a pathogenic virus infected with the human immune system cells. Since the discovery of the HIV virus, more than 25 million people have been killed since the discovery of the HIV virus, based on the detection of HIV (HIV) based on magnetic separation and chemiluminescence. 2 million 700 thousand new HIV infections are reported annually. Therefore, it is very important to study a new cheap and effective HIV virus diagnosis method. This study introduces a HIV detection analysis based on DNA hybridization and chemiluminescence. The one step reverse transcription polymerase chain reaction (RT-PCR) is used to amplify the HIV target sequence. The optimum annealing temperature is 58 C.B. Iotin-11-dUTP added into the one step RT-PCR amplification reaction, the magnetic beads modified by the Biotin. probe on the amplified products are used to capture these target sequences and generate the detection signals. This paper optimizes the different factors that may affect the chemiluminescence signal. Finally, the best hybridization temperature is produced at the concentration of the probe at 35 C.5 mu M. The best chemiluminescence signal. The best hybridization time is 30 minutes. This method shows a good specificity. When HCV and HBV infected serum replace HIV serum, the negative signal is obtained. The system successfully detects 10 copies of virus copy / milliliter of serum. Based on magnetic nanoparticles and chemiluminescence analysis, it extracts and detects many kinds of serum. Compared with the enzyme linked immunosorbent assay (ELISA) assay, the method based on nucleic acid detection is more sensitive. A variety of testing methods have been used to detect a variety of pathogens and can reduce the cost of nucleic acid detection. However, these methods can not reliably detect a large number of pathogens. In this study, a chemiluminescence detection based on DNA hybridization was established. The method, which is used to detect the number of pathogens at the same time, has no limitation on the number of pathogens. The probe has high specificity to capture virus sequences through the simultaneous extraction and detection of HBV, HCV and HIV. A single virus can be detected at the presence of higher loads in other viruses. The test can detect 10 copies / milliliters of blood. 10 copies / milliliters of HCV serum and 100 copies / milliliters of HIV serum. Based on magnetic separation technology from nucleic acid extraction to chemiluminescence detection, this method can be integrated into an automated system for convenient and high flux pathogen detection.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R446

【相似文献】