【作者】 孔建美；

【导师】 杨秀英； 马翠萍；

【作者基本信息】 青岛科技大学 ， 有机化学， 2010， 硕士

【摘要】 核酸适体(DNA aptamer)由于具有高亲和性及高特异性、分子量小、无免疫原性、配体广泛等优点,被越来越多的应用于目标分子(小分子或蛋白质)的识别和检测。而信号放大技术对不能进行直接扩增的低浓度待测分子的检测尤为重要,其中基于生物酶信号放大的荧光定量PCR技术和滚环复制(RCA)技术常用的体外扩增方法。在本论文的研究中,将核酸适体识别与生物酶信号放大的荧光分析法相结合,提出了一类新颖的、灵敏的可卡因的检测方法。其主要内容如下:1基于核酸适体和荧光定量PCR技术检测可卡因新方法的研究本章中,基于核酸适体识别与荧光定量PCR生物酶信号放大技术实现了可卡因的实时定量检测。为了实现低背景和高信噪比,本章利用磁珠分离技术实现了PCR模板链的彻底分离及富集,在很大程度上提高了检测方法的灵敏性。而荧光定量PCR技术对模板链进行了2n倍的扩增,更进一步地提高了检测方法的灵敏性。结合以上两种技术,该检测方法可检测到9 fmol级的可卡因,并显示出良好的特异性。在最佳检测条件下,可卡因浓度在10-10 mol/L ~ 10-6 mol/L之间时,PCR循环闭值Ct值与可卡因的浓度之间存在线性关系,检测限为3×10-11 mol/L。2基于核酸适体和滚环复制技术检测可卡因新方法的研究本章中,基于核酸适体识别及滚环复制生物酶信号放大技术,构建出一种高灵敏检测可卡因的荧光分析方法。当有目标物质可卡因存在时,可卡因与核酸适体A链上的序列特异性结合,将核酸适体互补链B链从A链上替换下来,再以替换下来的B链为引物环化探针C链,从而通过滚环复制技术实现可卡因的定量检测,检测限为20 pmol。DNA链也可以固定在芯片表面进行滚环复制扩增,但是芯片上的滚环复制产物很难实现定量检测,因此在均相中利用此技术进行信号放大对实现分析物的高灵敏定量检测变得尤为重要。另外,本实验方法还利用纳米金颗粒比表面积大,能够结合成百上千条DNA链的性质,提高检测方法的灵敏度。总之,本实验方法具有很高的灵敏度和良好的特异性,为复杂生物环境下物质的检测提供新的思路和模式。更多还原

【Abstract】 Aptamer has caught more and more attention in the target molecule (small molecule or protein) identification and testing because of the high affinity and specificity, low molecular weight, non-immunogenicity characteristics. And the signal amplification technology is particularly important to detect the molecules that can not be tested by direct amplification in a low concentration. In vitro,the nucleic acid amplification is one of the most important means of biotechnology research. The biologic enzyme-based signal amplification technologies such as fluorescence quantitative PCR (FQ-PCR) and rolling circle amplification (RCA) are the most commonly used signal amplification technology. In this study, the novel and sensitive detection method had been designed combining aptamer recognition with the biologic enzyme-based signal amplification. The major contents of the thesis were as follows:1 An aptamer-based and FQ-PCR amplification method for the detection of cocaineIn this chapter, a novel method based on aptamer identification and fluorescence quantitative PCR signal amplification technology for detection of cocaine was presented. In order to achieve low background and high signal/noise ratio, we used magnetic bead separation technology to achieve a complete separation and enrichment of PCR template chain. The sensitivity had been greatly improved. The template chain was amplified by 2n times by the fluorescence quantitative PCR technology, further improving the detection sensitivity. As a result of these two combined effects, 9 fmol level of cocaine could be detected using this method, and showed good specificity. In the optimum conditions, Ct exhibited a linear dependence on cocaine concentration in a wide range from 10-10 to 10-6 mol/L with a detection limit of 3×10-11 mol/L. 2 An aptamer-based and RCA amplification method for the detection of cocaineIn the present study, the method for highly sensitive detection of cocaine was developed based on aptamer identification and RCA signal amplification. When cocaine was in present, aptamer was specially combined with cocaine so that the supplementary B chain was replaced from aptamer. The B chain was directly used to cyclize probe C, and realized the quantitative detection of cocaine through the RCA technology. In this study, the detection limit of 20 pmol was achieved. When the DNA chain could also be adapted to surfaces of the chips, the quantitative RCA was proved difficult, further emphasizing the importance of real-time amplification methods for sensitive quantization in homogeneous phase solution. In addition, a single Au nanoparticle could be loaded with hundreds of DNA, which had greatly improved the sensitivity. In conclusion, this method exhibited excellent sensitivity, selectivity, good specificity, and provided a new idea and pattern for the molecular detection under complex biological environment.更多还原