肽核酸（PNA）探针检测鼠疫耶尔森氏菌的研究

发布时间：2018-03-18 03:35

本文选题：鼠疫菌　切入点：PNA　出处：《中国人民解放军军事医学科学院》2006年硕士论文　论文类型：学位论文

【摘要】：目的：利用特异的肽核酸(Peptide Nucleic Acid，PNA)探针、链霉亲和素包被的磁性纳米颗粒(磁珠)和Cy5纳米颗粒，结合荧光扫描技术，建立一种特异、快速、准确的检测鼠疫菌的方法。方法：针对鼠疫菌pMT1质粒上的caf1基因设计并合成一对特异PNA探针，经生物素标记后，分别与链霉亲和素包被的磁珠或Cy5纳米颗粒结合；探针与待测鼠疫菌的DNA杂交后，磁性纳米颗粒牢固连接靶核酸-探针结合体，用磁分离架收集后即可得纯化的特异核酸分子，经变性处理后对Cy5荧光纳米颗粒进行荧光扫描检测。本研究探讨了多个实验因素对测定的影响，并进行了特异性和灵敏度检测。此外，本研究将PNA探针与DNA探针、磁性纳米颗粒与市售磁珠、Cy5荧光纳米颗粒标记的探针与单个Cy5分子标记的探针进行比较，从而验证PNA探针、磁性纳米颗粒及Cy5荧光纳米颗粒在核酸检测技术中的优势。结果：利用Expedite 8909核酸合成系统合成了PNA探针，建立并优化了利用PNA探针检测鼠疫菌的方法，得到较好的线性关系(r=0.9914)；检测的灵敏度为O.9μg／mL(待测DNA)。通过多项比较，验证了PNA探针、磁性纳米颗粒及Cy5荧光纳米颗粒在核酸检测技术中的优势。结论：PNA探针的结构特点决定了其具有卓越的杂交亲和性能和特异识别能力，从而使本研究显示出较高的特异性和灵敏度，，具有较好的应用前景；磁性纳米颗粒的应用可简便且有效地将杂交复合体与未杂交核酸或过量的检测探针分离，大大降低非特异核酸分子的干扰，提高了检测灵敏度；Cy5荧光纳米颗粒具有的光稳定性和信号放大作用特点，使基于荧光纳米颗粒的荧光分析方法的灵敏度较传统方法显著提高。本研究建立的分析方法检测的灵敏度和特异性较高，能够灵敏、特异、稳定地对鼠疫菌进行定量检测，为鼠疫监控、诊断、应对紧急疫情的发生、动态的疫源地疫情监测提供了快速检测鉴定的有力手段。
[Abstract]:Objective: to establish a specific, rapid and accurate method for the detection of Yersinia pestis by using a specific peptide nucleic acid (PNA) probe, magnetic beads and Cy5 nanoparticles coated with streptavidin. Methods: a pair of specific PNA probes were designed and synthesized for the caf1 gene on the pMT1 plasmid of Yersinia pestis, which were labeled with biotin and bound to the magnetic beads or Cy5 nanoparticles coated with streptavidin, respectively, and hybridized with the DNA of Yersinia pestis. The magnetic nanoparticles are firmly connected to the target nucleic acid-probe, and the purified specific nucleic acid molecules can be obtained by magnetic separators. Cy5 fluorescent nanoparticles were detected by fluorescence scanning after denaturation. In this study, the effects of several experimental factors on the determination were investigated, and the specificity and sensitivity were detected. In addition, the PNA probe and the DNA probe were used in this study. The advantages of PNA probe, magnetic nanoparticles and Cy5 fluorescent nanoparticles in nucleic acid detection were verified by comparing the probe labeled with magnetic nanoparticles Cy5 fluorescent nanoparticles and those labeled by single Cy5 molecule in order to verify the advantages of PNA probes, magnetic nanoparticles and Cy5 fluorescent nanoparticles in nucleic acid detection. Results: the PNA probe was synthesized by using Expedite 8909 nucleic acid synthesis system, and the method of detecting Yersinia pestis with PNA probe was established and optimized. A good linear relationship was obtained, and the sensitivity of detection was 0. 9 渭 g / mL. Advantages of magnetic nanoparticles and Cy5 fluorescent nanoparticles in nucleic acid detection. Conclusion the structure of the probe determines that it has excellent hybridization affinity and specific recognition ability, which makes this study show high specificity and sensitivity, and has a good prospect of application. The magnetic nanoparticles can be used to separate the hybrid complex from non-hybrid nucleic acid or excessive detection probe, which greatly reduces the interference of non-specific nucleic acid molecules. The detection sensitivity of Cy5 fluorescent nanoparticles is improved, which has the characteristics of optical stability and signal amplification. The sensitivity of the fluorescent analysis method based on fluorescent nanoparticles was significantly higher than that of the traditional method. The sensitivity and specificity of the analytical method established in this study were higher than that of the traditional method. The method was able to detect Yersinia pestis quantitatively with sensitivity, specificity and stability. It provides a powerful means for rapid detection and identification of plague surveillance, diagnosis, emergency outbreak and dynamic foci surveillance.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R378

【参考文献】