基于辅助探针与免酶的扩增技术对病毒DNA的检测

发布时间：2018-10-12 19:12

【摘要】：HIV病毒和HBV DNA病毒是当下研究的热点。因其对人类造成摧毁性的害处而闻名于世。DNA是储存、复制和传递遗传信息的主要物质。检测病毒的DNA对病毒的感染判定、病程监控,以及探究疾病内源有至关重要的作用。因此,建立一种快速,有效,简便的检测病毒DNA的方法非常重要。荧光法以其快速,简便,高效等优点被广泛应用于核酸检测。信号放大技术是非常实用的技术,能够使检测变得更加灵敏。同时无酶无标记法能够减低实验成本,是实验变得更加快速。本文还采用了辅助DNA探针进行链替换反应,省去了设计多条分子信标的麻烦。使的本实验更加简便,有效。1、设计了一个发卡型分子信标MB和两条辅助探针。借助G-四联体为信号显示平台。当目标物DNA加入时,MB与目标物进行杂交反应。随后辅助探针与他们的杂交产物进行链取代反应,被取代下来的DNA继续和其他MB进行反应,进行循环扩增。选用THT染料(硫代黄色素)与G-四联体结合,我们通过荧光分光光度计能检测到信号增强了3倍。在目标物浓度范围为(0.1 nM-50 nM)时,取得一个良好的线性效果。检出限位50 pM。2、设计修饰了二氨基嘌呤的发卡结构分子信标MB.利用二氨基嘌呤在单链DNA上显示荧光,双链上不显示荧光的特点。加入目标物与分子信标进行杂交链反应,荧光法检测到2氨基嘌呤释放荧光。当用辅助探针将目标物替换下来后,目标物继续与分子信标MB反应,产生大量的能够释放二氨基嘌呤产物。信号扩增了3.4倍。无需荧光猝灭。检出限为80 pM。3、设计了末端能够形成G-四联体结构的分子信标H1以及另外一条分子信标H2。NMM(N-甲基卟啉二丙酸IX)与其结合产生一定荧光信号。目标物与分子信标H1的结合使分子信标打开,G-四联体结构不能形成。荧光下降。H2与其杂交产物进行链取代反应,以致目标物循环。荧光下降4倍。检出限为65 pM。
[Abstract]:HIV virus and HBV DNA virus are the focus of current research. Known for its devastating effects on humans, DNA is the main material for storing, copying, and transmitting genetic information. Detection of virus DNA plays an important role in virus infection detection, disease course monitoring, and disease endogenous detection. Therefore, it is very important to establish a rapid, effective and simple method for the detection of viral DNA. Fluorescence method has been widely used in nucleic acid detection for its advantages of fast, simple and high efficiency. Signal amplification is a very practical technique, which can make detection more sensitive. At the same time, enzyme-free labeling can reduce the cost of the experiment, so the experiment becomes faster. An auxiliary DNA probe is also used for chain substitution reaction, which saves the trouble of designing multiple molecular beacons. This experiment is more simple and effective. 1. A card type molecular beacon MB and two auxiliary probes are designed. G-quadruple is used as signal display platform. When the target DNA was added, the MB was hybridized with the target. Then the auxiliary probes reacted with their hybridization products and the replaced DNA continued to react with other MB for cyclic amplification. By using THT dye (thioyellow pigment) combined with G-quadruple, we can detect the signal by three times by fluorescence spectrophotometer. A good linear effect was obtained when the concentration of the target was (0. 1 nM-50 nM). The detection limit of 50 pM.2, was designed to modify the molecular beacon MB. of diaminopurine. Using diaminopurine to display fluorescence on single strand DNA, no fluorescence on double strand. The 2 aminopurine was detected by fluorescence method in the hybridization chain reaction between the target and the molecular beacon. When the target was replaced by an auxiliary probe, the target continued to react with the molecular beacon MB to produce a large amount of diaminopurine products. The signal amplification was 3.4 times. There is no need for fluorescence quenching. The detection limit of 80 pM.3, was used to design a molecular beacon H1 which could form a G-quadruplex structure and another molecular beacon H2.NMM (N-methylporphyrin dipropionate IX) to produce a certain fluorescence signal. The binding of the target and the molecular beacon H 1 makes the molecular beacon open and the G- quadruple structure cannot be formed. The fluorescence of H _ 2 was decreased. The H _ 2 chain substitution reaction was carried out with its hybrid product, which resulted in the circulation of the target material. The fluorescence decreased by four times. Detection limit is 65 pM.
【学位授予单位】：湘潭大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.6

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