多序列比对的量子点荧光探针检测金黄色葡萄球菌的研究

Detection of Staphyloccocus Aureus 16S rDNA Using QD FRET Bioprobe Based on Multiple Sequence Alignment

利用以量子点(Quantum dot,QD)作为供体、有机荧光染料作为受体的荧光能量共振转移(Fluorescenceresonance energy transfer,FRET)体系检测核酸等大分子是一种非常重要的检测手段。本文构建了一种检测金黄色葡萄球菌种特异性16S rDNA的新方法。此方法以羧基修饰的525 nm量子点与氨基修饰的DNA在EDC的作用下通过脱水连接形成QD-DNA复合物作为荧光能量共振转移体系的供体、有机荧光基团ROX修饰的DNA作为荧光能量共振转移体系的受体组成能与金黄色葡萄球菌种特异性16S rDNA杂交的检测探针。当探针与靶序列发生杂交时,作为供体的525 nm QD与作为受体的ROX之间的距离被缩短至能有效发生荧光能量共振转移的距离之内。此时,以不能致ROX发光的波长激发量子点发光,其荧光强度下降,而ROX的荧光强度上升。在不存在靶序列的情况下,不会发生这种荧光强度的变化。QD与ROX荧光强度的变化是实现本检测体系快速、简单的重要保证。

Fluorescence resonance energy transfer （FRET） that consists of quantum dot as donors and organic fluorophore dyes as acceptors has been a very important method to detect biomolecules such as nucleic acids. In this work, we established a new FRET detection system of Staphyloccocus aureus species-specific 16S rDNA using QD-ROX FRET bioprobe, in which 525 nm QD-DNA conjugation consisted of the carboxyl-modified QD and the amino-modified DNA in the presence of EDC. Both ROX-DNA and the conjugation above could hybridize with the target DNA after forming the QD- ROX bioprobe. When the hybridization made the distance between the QD and ROX to meet FRET effect needed, 525 nm QD fluorescence intensity decreased and ROX fluorescence intensity increased. In the control, there was no notable change of fluorescence intensities without target DNA. It is very clear that the change of the QD and ROX fluorescence intensities provide the good base and guaranty for this rapid and simple detection system.