基于切刻酶的等温核酸扩增检测新技术的研究

发布时间：2018-02-01 18:40

本文关键词： 等温扩增信号放大分子信标核酸检测 POCT　出处：《青岛科技大学》2016年硕士论文　论文类型：学位论文

【摘要】：核酸作为重要的遗传信息生物分子,其检测被广泛的应用在食品安全、生物医学检测、环境检测等诸多领域。聚合酶链式反应(Polymerase Chain Reaction,PCR)是目前应用最广泛的核酸扩增检测方法之一,然而PCR技术需要专门的热循环仪,限制了其在疾病的即时诊断等领域的应用。因此,发展更灵敏、快速的核酸等温检测方法具有重要意义。本论文构建了几种简单、快速的核酸等温扩增检测新方法,分别对DNA和RNA进行了快速、灵敏和特异地检测。在第二章中,本论文建立了一种基于双切口分子信标的等温扩增检测DNA新方法的研究。用该方法对提取的大肠杆菌16S r DNA进行实时荧光检测,当检测靶标的量在100 fmol到10 amol之间时呈良好的线性,最低检测限为10 amol;通过在引物上设计突变碱基进行检测,验证了该方法具有较强的特异性;并用该方法在复杂体系中检测时呈现较强的抗干扰能力。此外,它是一锅式的等温链置换扩增方法,不需要额外的操作步骤,极大的简化了实验步骤,并降低了样品污染的可能性。它的这些优点使该方法在核酸的现场快速检测及即时检测(Point-of-care testing,POCT)中更具有实用性。目前的RNA检测方法中,大部分都需要利用反转录酶将RNA反转录为c DNA后再进行扩增检测。本论文第三章构建了三种无需加反转录酶可直接等温扩增RNA的实验方案,实现了对RNA的快速“一步法”检测。这几种方法都利用了本实验室发现的Bst DNA聚合酶既具有DNA聚合酶活性又具有内在的反转录酶活性的特点;此外,还充分的利用了DNA聚合酶的链置换活性及与切刻酶联用的扩增放大机理,最终实现了信号的多重放大。最优方法对大肠杆菌16S r DNA的检测可达1 amol,对大肠杆菌16S r RNA在一定的浓度下也可以进行有效的检测。这些方法均具有在等温、均相条件下可直接检测RNA。在RNA的检测中大大缩短反应时间,简化操作步骤,降低实验成本和操作难度。这些新的等温方法的建立为其他核酸检测提供了新方法与新思路,同时对即时检测也具有重要意义。
[Abstract]:Nucleic acid as an important genetic information biomolecules, its detection is widely used in food safety, biomedical detection. Polymerase chain reaction (PCR) is one of the most widely used nucleic acid amplification methods. However, PCR technology needs specialized thermal circulator, which limits its application in the field of disease diagnosis and so on. Therefore, it is more sensitive to develop. Rapid isothermal detection of nucleic acid is of great significance. In this paper, a few simple and fast methods of nucleic acid isothermal amplification were constructed, and DNA and RNA were used for rapid detection. Sensitive and specific detection. In Chapter II. In this paper, a new method of isothermal amplification for detection of DNA based on double cut molecular beacon was established. The method was used to detect 16s r DNA of Escherichia coli in real time. When the amount of target is between 100 fmol and 10 amol, the detection limit is 10 amol. By designing the mutation base on the primer for detection, it is proved that the method has strong specificity. In addition, it is a one-pot isothermal chain replacement amplification method, which does not require additional operation steps, greatly simplifies the experimental steps. These advantages enable the method to detect nucleic acid quickly in situ and to detect Point-of-care testing in real time. In current RNA detection methods. Most of them need to use reverse transcriptase to reverse RNA to c DNA before amplification detection. In the third chapter of this paper, three kinds of RNA can be directly isothermal amplified without reverse transcriptase. The fast "one step" detection of RNA is realized. All of these methods make use of the Bst found in our laboratory. DNA polymerase has both DNA polymerase activity and intrinsic reverse transcriptase activity. In addition, the chain replacement activity of DNA polymerase and the amplification and amplification mechanism of DNA polymerase combined with cutting enzyme were also fully utilized. Finally, the multiple amplification of the signal was realized, and the detection of 16s r DNA of E. coli by the optimal method was up to 1 amol. 16s r RNA of Escherichia coli can also be effectively detected at a certain concentration. RNA can be directly detected under homogeneous conditions. In the detection of RNA, the reaction time is greatly shortened and the operation procedure is simplified. The establishment of these new isothermal methods provides new methods and new ideas for other nucleic acid detection, and is also of great significance for real-time detection.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.3

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