Department of Neurology, The First Affiliated Hospital, Jinan University, Guangzhou 510630, PR China; Institute of Pharmaceutical Analysis, College of Pharmacy/State Key Laboratory of Bioactive Molecules and Druggability Assessment/Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research of China, Jinan University, Guangzhou 510632, PR China; School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, PR China.
Institute of Pharmaceutical Analysis, College of Pharmacy/State Key Laboratory of Bioactive Molecules and Druggability Assessment/Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research of China, Jinan University, Guangzhou 510632, PR China.
Biosens Bioelectron. 2024 Nov 15;264:116641. doi: 10.1016/j.bios.2024.116641. Epub 2024 Aug 8.
Rapid and accurate diagnosis of tuberculosis (TB) is of great significance to control the spread of this devastating infectious disease. In this work, a sensitive and low-cost point-of-care testing (POCT) detection platform for TB was developed based on recombinase polymerase amplification (RPA)-catalytic hairpin assembly (CHA)-assisted dual signal amplification strategy. This platform could achieve homogeneous fluorescent and visual diagnosis of TB by using CdTe quantum dots (QDs) signal reporter. In the presence of target DNA (IS1081 gene fragment), RPA amplicons blocked by short oligonucleotide strands could trigger CHA signal amplification, leading to the Ag releasing from C-Ag-C structure and the fluorescence quenching of CdTe QDs by the released Ag. Furthermore, the detection performance of CdTe QDs modified by 3-mercaptopropionic acid (MPA) or thiomalic acid (TMA) (MPA-capped QDs and TMA-capped QDs) was systematically compared. Experimental results demonstrated that TMA-capped QDs exhibited better detection sensitivity due to their stronger interaction with Ag. The limits of detection (LODs) of fluorescence and visual analysis were as low as 0.13 amol L and 0.33 amol L. This method was successfully applied to the clinical sputum samples from 36 TB patients and 20 healthy individuals, and its quantitative results were highly consistent with those obtained by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). The proposed approach has the advantages of high sensitivity and specificity, simple operation and low cost, and is expected to be applied in clinical TB screening and diagnosis.
快速准确地诊断结核病（TB）对于控制这种毁灭性传染病的传播具有重要意义。在这项工作中，基于重组酶聚合酶扩增（RPA）-催化发夹组装（CHA）辅助的双信号放大策略，开发了一种用于 TB 的灵敏且低成本的即时护理检测（POCT）检测平台。该平台可以通过使用碲化镉量子点（QDs）信号报告器实现 TB 的均相荧光和可视化诊断。在存在靶 DNA（IS1081 基因片段）的情况下，被短寡核苷酸链阻断的 RPA 扩增子可以触发 CHA 信号放大，导致 Ag 从 C-Ag-C 结构中释放出来，并且被释放的 Ag 使 CdTe QDs 的荧光猝灭。此外，还系统比较了巯基丙酸（MPA）或硫代马来酸（TMA）修饰的 CdTe QDs（MPA 封端的 QDs 和 TMA 封端的 QDs）的检测性能。实验结果表明，由于与 Ag 的相互作用更强，TMA 封端的 QDs 表现出更好的检测灵敏度。荧光和目视分析的检出限（LOD）低至 0.13 amol L 和 0.33 amol L。该方法成功应用于 36 例 TB 患者和 20 例健康个体的临床痰液样本，其定量结果与实时荧光定量聚合酶链反应（RT-qPCR）的结果高度一致。该方法具有灵敏度高、特异性强、操作简单、成本低等优点，有望应用于临床 TB 筛查和诊断。
