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利用适配体的识别能力和可扩增性,构建了基于微磁珠分离技术的适配体实时定量聚合酶链式反应(PCR)检测方法.通过微磁珠偶联的互补链与适配体序列之间的碱基配对结合,有效除去溶液中未与靶分子结合的适配体序列,采用实时定量PCR技术测定上清液中结合态的适配体序列浓度,从而间接实现对靶分子的定量检测.分别选取代表生物大分子和有机小分子的凝血酶和ATP作为检测对象,验证了该方法的普适性.研究结果表明,在获取特异性适配体序列后,仅需简单优化其互补链序列,即可对超低含量的凝血酶和ATP进行准确定量,检出限分别为50 pmol/L和5μmol/L.该方法具有同时适用于高特异性和高灵敏度地检测生物大分子和有机小分子的优势.
Based on the recognition ability and expandability of aptamers, a real-time quantitative polymerase chain reaction (PCR) detection method based on micro-magnetic beads separation technology was established. Base sequence pairwise binding, effective removal of the solution is not the target molecule binding aptamer sequence, using real-time quantitative PCR technology to determine the bound supernatant aptamer sequence concentration, and thus indirect realization of the target molecule The thrombin and ATP, which represent biological macromolecules and small organic molecules, respectively, were selected as test objects to verify the universality of this method.The results show that after obtaining specific aptamer sequences, only simple optimization Its complementary strand sequence can accurately determine ultra-low levels of thrombin and ATP with the detection limits of 50 pmol / L and 5 μ mol / L. The method has the advantages of being suitable for both high specificity and high sensitivity for detecting large organisms Advantages of molecules and small organic molecules.