Kapadia Jay Bhakti, Daoud Jamal, Perreault Jonathan
INRS-Armand Frappier Institute-531, Boul. Des Prairies, Laval, QC, H7 V 1B7, Canada.
Galenvs Sciences-6750 Rue Hutchison, Montreal, QC, H3N 1Y4, Canada.
Analyst. 2025 May 12;150(10):2019-2028. doi: 10.1039/d4an01212g.
Toehold mediated strand displacement reaction (TMSDR) offers a rapid, enzyme-free amplification strategy, providing advantages over traditional methods like RT-PCR, and RT-LAMP. Optimizing TMSDR can significantly enhance sensitivity in point-of-care biosensor applications for target nucleic acid detection. However, achieving optimal performance requires meticulous probe design and stringent quality control. We developed a TMSDR-based system targeting a specific SARS-CoV-2 RNA sequence through testing multiple fluorophore-quencher labeled DNA probes. Following optimization, a probe with a strategically designed: stem, loop, and optimized toehold length emerged as the most effective candidate. Displacer sequence optimization further enhanced amplification efficiency. Ensuring probe purity is crucial, as impurities elevated background noise and diminished sensitivity. This work underscores the importance of rigorous probe quality in achieving reliable and sensitive TMSDR-based viral RNA detection, paving the way for robust point-of-care diagnostic tools.
引发链介导的链置换反应（TMSDR）提供了一种快速、无酶的扩增策略，比传统方法如逆转录聚合酶链反应（RT-PCR）和逆转录环介导等温扩增技术（RT-LAMP）更具优势。优化TMSDR可以显著提高即时检测生物传感器在目标核酸检测应用中的灵敏度。然而，要实现最佳性能需要精心设计探针并进行严格的质量控制。我们通过测试多种荧光团-淬灭剂标记的DNA探针，开发了一种基于TMSDR的系统，该系统靶向特定的严重急性呼吸综合征冠状病毒2（SARS-CoV-2）RNA序列。经过优化，一种具有精心设计的茎、环和优化的引发链长度的探针成为最有效的候选者。置换序列优化进一步提高了扩增效率。确保探针纯度至关重要，因为杂质会增加背景噪音并降低灵敏度。这项工作强调了严格的探针质量对于实现基于TMSDR的可靠且灵敏的病毒RNA检测的重要性，为强大的即时诊断工具铺平了道路。
