Department of Chemistry, University of Toronto, 80. St. George Street, Toronto, Ontario, M5S 3H6, Canada.
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario, M5S 3E1, Canada.
Lab Chip. 2023 Dec 20;24(1):63-73. doi: 10.1039/d3lc00683b.
There is great enthusiasm for using loop-mediated isothermal amplification (LAMP) in point-of-care nucleic acid amplification tests (POC NAATs), as an alternative to PCR. While isothermal amplification techniques like LAMP eliminate the need for rapid temperature cycling in a portable format, these systems are still plagued by requirements for dedicated optical detection apparatus for analysis and manual off-chip sample processing. Here, we developed a new microfluidic system for LAMP-based POC NAATs to address these limitations. The new system combines digital microfluidics (DMF) with distance-based detection (DBD) for direct signal readout. This is the first report of the use of (i) LAMP or (ii) DMF with DBD - thus, we describe a number of characterization steps taken to determine optimal combinations of reagents, materials, and processes for reliable operation. For example, DBD was found to be quite sensitive to background signals from low molecular weight LAMP products; thus, a Capto™ adhere bead-based clean-up procedure was developed to isolate the desirable high-molecular-weight products for analysis. The new method was validated by application to detection of SARS-CoV-2 in saliva. The method was able to distinguish between saliva containing no virus, saliva containing a low viral load (10 genome copies per mL), and saliva containing a high viral load (10 copies per mL), all in an automated system that does not require detection apparatus for analysis. We propose that the combination of DMF with distance-based detection may be a powerful one for implementing a variety of POC NAATs or for other applications in the future.
人们热衷于在即时检验核酸扩增检测（POC NAAT）中使用环介导等温扩增（LAMP），将其作为 PCR 的替代方法。虽然等温扩增技术（如 LAMP）消除了在便携式设备中快速温度循环的需求，但这些系统仍然需要专用的光学检测设备进行分析和手动的离片样本处理。在这里，我们开发了一种新的基于 LAMP 的即时检验 POCT 系统，以解决这些限制。该新系统将数字微流控（DMF）与基于距离的检测（DBD）相结合，用于直接信号读出。这是首次报道使用（i）LAMP 或（ii）DMF 与 DBD - 因此，我们描述了为了实现可靠操作，确定最佳试剂、材料和工艺组合所采取的一些特征描述步骤。例如，DBD 对低分子量 LAMP 产物的背景信号非常敏感；因此，开发了 Capto™粘附珠基清洗程序，以分离用于分析的理想高分子量产物。该新方法通过应用于唾液中 SARS-CoV-2 的检测得到了验证。该方法能够区分不含病毒的唾液、含有低病毒载量（10 个基因组拷贝/mL）的唾液和含有高病毒载量（10 拷贝/mL）的唾液，所有这些都在一个不需要分析检测设备的自动化系统中完成。我们提出，DMF 与基于距离的检测相结合可能是实现各种即时检验 NAAT 或未来其他应用的强大方法。
