Department of Chemistry, Iowa State University, Ames, IA, 50011, USA.
Department of Chemistry, Iowa State University, Ames, IA, 50011, USA.
Anal Chim Acta. 2019 Jan 3;1045:132-140. doi: 10.1016/j.aca.2018.09.014. Epub 2018 Sep 12.
Isothermal nucleic acid amplification (INAA) techniques such as loop-mediated isothermal amplification (LAMP) and isothermal multiple-self-matching-initiated amplification (IMSA) constitute simple and rapid approaches for the detection of pathogens. However, due to the employment of multiple primers, the detection of LAMP and IMSA products is easily influenced by high background signals from primer dimer-based nonspecific nucleic acid amplification (NSA) products. Moreover, time-consuming sample preparation steps are often required for the isolation of sufficiently pure nucleic acid prior to INAA. To address these drawbacks, hydrophobic magnetic ionic liquids (MILs) were used to rapidly preconcentrate DNA from complex biological samples followed by direct amplification by LAMP and IMSA. Careful control of the components within the isothermal buffer permitted direct addition of DNA-enriched MIL to the INAA reaction mixture, thereby circumventing tedious purification procedures that are ordinarily required prior to downstream DNA amplification. When added directly to INAA reactions, MIL solvents released metal ions that ultimately inhibited the primer dimer-mediated NSA, resulting in a flat or decreased baseline signal in no-template control samples and short threshold time for positive reactions. Using a MIL-based single droplet DNA extraction method, MIL-enhanced INAA reaction system, and a handheld 3D printed device for visual detection of the amplified product in customized tubes, we demonstrate the potential of the MIL-based approach for the onsite analysis of DNA from pathogens.
等温核酸扩增（INAA）技术，如环介导等温扩增（LAMP）和等温多自我匹配引发扩增（IMSA），构成了检测病原体的简单快速方法。然而，由于使用了多个引物，LAMP 和 IMSA 产物的检测容易受到基于引物二聚体的非特异性核酸扩增（NSA）产物的高背景信号的影响。此外，在进行 INAA 之前，通常需要耗时的样品制备步骤来分离足够纯的核酸。为了解决这些缺点，疏水性磁性离子液体（MILs）被用于从复杂的生物样品中快速浓缩 DNA，然后直接通过 LAMP 和 IMSA 进行扩增。通过对等温缓冲液中的成分进行仔细控制，可以将富含 DNA 的 MIL 直接添加到 INAA 反应混合物中，从而避免了通常在下游 DNA 扩增之前需要进行的繁琐的纯化步骤。当直接添加到 INAA 反应中时，MIL 溶剂会释放出金属离子，最终抑制引物二聚体介导的 NSA，导致无模板对照样品中的基线信号平坦或降低，以及阳性反应的阈值时间缩短。我们使用基于 MIL 的单个液滴 DNA 提取方法、MIL 增强的 INAA 反应系统以及用于在定制管中可视化检测扩增产物的手持式 3D 打印设备，展示了基于 MIL 的方法在现场分析病原体 DNA 方面的潜力。
