基于磁分离和荧光量子点标记的高灵敏禽流感病毒检测

发布时间：2018-09-08 12:15

【摘要】：高灵敏的病毒检测在食品安全,疾病防控与治疗,以及生物安全等方面均至关重要。随着科学技术的进步,各种检测方法得到了大大地发展,但是目前仍缺乏适合现场运用的高灵敏的检测方法。以禽流感病毒为例,自禽流感病毒被发现以来已经造成多次世界范围内禽类或者人类疑似流感的大流行。目前,禽流感病毒检测的标准方法是在鸡胚或者细胞中对病毒进行分离扩增,并使用血清学方法确认病毒类型,该方法步骤繁琐,一般需要1-2周的时间才能得到结果,不能实现快速的现场检测。其他常用的检测方法有酶联免疫吸附方法(ELISAs)和基于分子生物学的核酸扩增方法(PCR)。由于临床样品成分复杂和待检测物质浓度低,临床样品的检测对检测方法的灵敏度、稳定性、可靠性有着很高的要求；同时,面向现场的临床样品的检测要求检测方法操作简便、便携性强、无需复杂仪器,因此,酶联免疫方法(ELISA)和常规的PCR方法还无法实现面向现场的高灵敏、稳健的检测。基于微/纳米磁球的免疫磁分选方法能将低浓度的目标物从复杂的样品环境中识别和分离,降低非特异性物质的干扰；同时,利用荧光纳米材料量子点的稳定高亮的荧光,使用量子点作为标记材料可以提高检测的灵敏度。将微/纳米磁球的磁分选与荧光量子点标记结合起来的方法在高灵敏病原体检测中有着很好的运用前景；并且由于该方法具有操作简便、原理普适、无需人员操作培训等优点,该方法有望实现在现场以及临床病毒检测。因此,本论文致力于构建可运用于实际样品的现场检测的稳健方法,将微/纳米磁球的磁分选与荧光量子点标记结合起来用于禽流感病毒H9N2高灵敏、稳定的检测。本论文主要完成了以下工作： 1.以禽流感病毒H9N2为例,构建了一种基于免疫磁分离和荧光量子点标记的稳健的可运用于现场的高灵敏禽流感病毒检测方法。该方法通过在纳米磁球表面修饰抗H9N2膜蛋白HA的单克隆抗体,实现了即使在复杂环境中H9N2病毒的准确捕获和分离；利用免疫夹心策略,通过生物素化的抗体引入偶联了链酶亲和素的荧光半导体材料量子点,实现对目标病毒高灵敏的检测。该方法特异性强、灵敏度高,可实现200μL样品中60个病毒的检测,是目前最灵敏的病毒检测方法之一。同时,该方法具有很好的可重复性和稳定性,其批次内变异系数为1.35%,批次间变异系数为3.30%。并且,该方法可高选择性的检测复杂生物样品(破碎的组织和禽类粪便)中的病毒。30个禽类咽拭子临床样品的双盲测试验证结果符合率高达96.7%,说明该方法具有很好的可靠性。因此,该方法在实际样品的现场检测中有着广阔的运用前途。并且由于其普适的检测原理,该检测方法可推广至其他病毒的检测。 2.研究了纳米磁球捕获病毒过程的动力学。在这个体系中,用于捕获的纳米级别免疫磁球与病毒有着相当的尺寸(100-200nm),因此研究免疫磁球捕获病毒过程的动力学有助于了解纳米级别的颗粒之间的反应动力学。我们发现,在免疫磁球大大过量的情况下,该捕获过程类似于双分子准一级反应,其捕获速率常数kf为4.25×109(mol/L)-1s-1,且捕获90%的病毒仅需15分钟。 3.从禽流感病毒感染宿主细胞时病毒识别和结合的细胞表面a-2,3-唾液酸-半乳糖酸受体(α-2,3sialic acid-Gal linkage)受到启发,在特定唾液酸酶的作用下,将唾液酸通过α-2,3糖苷键连接到偶联了半乳糖的量子点的表面以模拟细胞表面唾液酸受体结构,成功制备唾液酸修饰的量子点,并尝试对禽流感病毒H9N2进行标记。 4.基于免疫磁分选和荧光量子点标记高灵敏检测病毒方法的原理,在禽流感病毒被磁球捕获后,初步建立了利用修饰了a-2,3唾液酸的量子点(α-2,3-SA-QDs)对被捕获的禽流感病毒进行识别并检测的新方法。
[Abstract]:With the development of science and technology, a variety of detection methods have been greatly developed, but there is still a lack of highly sensitive detection methods suitable for field use. At present, the standard method of avian influenza virus detection is to isolate and amplify the virus in chicken embryos or cells, and use serological methods to confirm the type of the virus. This method is cumbersome and usually takes 1-2 weeks to get the results. Other commonly used detection methods are enzyme-linked immunosorbent assay (ELISAs) and DNA amplification based on molecular biology (PCR). Because of the complexity of clinical samples and the low concentration of the substances to be detected, the detection of clinical samples requires high sensitivity, stability and reliability of detection methods. In order to detect clinical samples on the spot, it is required that the method is simple, portable and does not need complicated instruments. Therefore, enzyme-linked immunosorbent assay (ELISA) and conventional PCR methods can not achieve high sensitivity and robustness on the spot.
Immunomagnetic separation method based on micro/nano magnetic spheres can identify and separate low concentration targets from complex sample environments and reduce the interference of non-specific substances. At the same time, using stable and bright fluorescence of fluorescent nano-material quantum dots, using quantum dots as marker materials can improve the detection sensitivity. The combination of sphere magnetic sorting and fluorescent quantum dot labeling has a good application prospect in the detection of highly sensitive pathogens; and because this method has the advantages of simple operation, universal principle, no need for personnel training, this method is expected to achieve in-situ and clinical virus detection. A robust method for in-situ detection of avian influenza virus H9N2 was developed by combining magnetic separation of micro/nano magnetic spheres with fluorescent quantum dot labeling.
1. Taking H9N2 avian influenza virus as an example, a robust and in-situ detection method for avian influenza virus based on immunomagnetic separation and fluorescent quantum dot labeling was constructed. The method can capture H9N2 virus accurately even in complex environment by modifying monoclonal antibodies against H9N2 membrane protein HA on the surface of nano-magnetic spheres. The method has strong specificity and high sensitivity, and can detect 60 viruses in 200 mu L samples. It is one of the most sensitive methods to detect viruses. The intra-batch and inter-batch variability coefficients were 1.35% and 3.30%, respectively. The method can be used to detect virus in complex biological samples (broken tissues and poultry feces) with high selectivity. The coincidence rate of double-blind test was 96.7% in 30 poultry pharyngeal swabs. Therefore, this method has a broad application prospect in the field detection of real samples. And because of its universal detection principle, this method can be extended to other virus detection.
2. The kinetics of virus capture by nano-magnetic spheres was studied. In this system, the nano-sized immunomagnetic spheres used for capture have the same size as the virus (100-200 nm). Therefore, the kinetics of virus capture by immunomagnetic spheres is helpful to understand the reaction kinetics between nano-sized particles. In the case of large excess, the capture process is similar to the bimolecular quasi-first order reaction. The capture rate constant KF is 4.25 *109 (mol/L) -1s-1, and it takes only 15 minutes to capture 90% of the virus.
3. Inspired by the virus's recognition and binding of a-2,3-sialic acid-galactic acid receptor (a-2,3-sialic acid-Gal linkage) on the surface of cells infected with avian influenza virus, sialic acid was linked to the surface of quantum dots coupled with galactose via a-2,3-glycoside bond under the action of a specific sialic enzyme to simulate saliva on the cell surface. The acid receptor structure has successfully prepared sialic acid modified quantum dots and attempted to mark the avian influenza virus H9N2.
4. Based on the principle of immunomagnetic sorting and fluorescent quantum dot labeling, a new method for the identification and detection of avian influenza virus was established by using a-2,3-SA-QDs modified with a-2,3-sialic acid (a-2,3-SA-QDs).
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392.1;O657.1

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