基于微磁珠分离技术的适配体实时定量PCR检测方法

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5 μmol/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.

Establishment and Application of a Magnetic Separation-based Aptameric Real-time Quantitative PCR Detection Approach

Aptamers, as recognition molecules, have been applied in a variety of fields due to their well-known advantages. However, there is still a big difficulty to establish a universal aptameric assay in the field of analytical chemistry. In this research, benefited from the specific recognition and the amplification capability of aptamer, a magnetic separation-based aptameric real-time quantitative PCR detection approach was demonstrated. A complementary sequence immobilized onto magnetic beads was applied to effectively remove the free aptamers which did not interact with target molecules in solution by virtue of base-pair binding capability between the complementary sequence and the aptamer sequence. After being separated under magnetic field, the concentrations of aptamers corresponding to various concentrations of target molecules were collected from supernatant solution, and determined by real-time quantitative PCR analysis. To demonstrate the universality of such an approach, thrombin and ATP were chosen as detection targets for representing biomacromolecules and organic small molecules, respectively. The experimental results indicated that the ultra low concentrations of thrombin and ATP spiked in binding buffer could be successfully quantified only after a simple optimization of complementary sequences and other conditions, their limit of detections(LODs) were down to 50 pmol/L and 5 μmol/L, respectively. Currently, this approach was one of the few aptameric assays which could be applied to determine biomacromolecules and organic small molecules with high specificity and sensitivity.