Multiplex Detection of Nucleic Acids Using Recombinase Polymerase Amplification and a Molecular Colorimetric 7-Segment Display,ACS Omega

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Multiplex Detection of Nucleic Acids Using Recombinase Polymerase Amplification and a Molecular Colorimetric 7-Segment Display
ACS Omega ( IF 3.7 ) Pub Date : 2019-07-01 00:00:00 , DOI: 10.1021/acsomega.9b01097
Jia Li ₁ , Nina M. Pollak _{1,

2} , Joanne Macdonald _{1,

3}

Affiliation

Nucleic acid analysis has become highly relevant for point-of-care (POC) diagnostics since the advent of isothermal amplification methods that do not require thermal cycling. In particular, recombinase polymerase amplification (RPA) combined with lateral flow detection offers a rapid and simple solution for field-amenable low-resource nucleic acid testing. Expanding POC nucleic acid tests for the detection of multiple analytes is vital to improve diagnostic efficiency because increased multiplexing capacity enables higher information density combined with reduced assay time and costs. Here, we investigate expanding RPA POC detection by identifying a generic multiplex RPA format that can be combined with a generic multiplex lateral flow device (LFD) to enable binary and molecular encoding for the compaction of diagnostic data. This new technology relies on the incorporation of molecular labels to differentiate nucleic acid species spatially on a lateral flow membrane. In particular, we identified additional five molecular labels that can be incorporated during the RPA reaction for subsequent coupling with LFD detection. Combined with two previously demonstrated successful labels, we demonstrate potential to enable hepta-plex detection of RPA reactions coupled to multiplex LFD detection. When this hepta-plex detection is combined with binary and molecular encoding, an intuitive 7-segment output display can be produced. We note that in all experiments, we used an identical DNA template, except for the 5′ label on the forward primer, to eliminate any effects of nucleic acid sequence amplification bias. Our proof-of-concept technology demonstration is highly relevant for developing information-compact POC diagnostics where space and time are premium commodities.

中文翻译：

使用重组酶聚合酶扩增和分子比色7段显示器对核酸进行多重检测

自从不需要热循环的等温扩增方法问世以来，核酸分析已与即时诊断（POC）诊断高度相关。特别是重组酶聚合酶扩增（RPA）与侧向流检测相结合，为现场可适应的低资源核酸测试提供了一种快速，简单的解决方案。扩展POC核酸测试以检测多种分析物对于提高诊断效率至关重要，因为增加的多路复用能力可实现更高的信息密度，同时减少测定时间和成本。在这里，我们研究通过识别可与通用多路复用侧向流动设备（LFD）结合使用的通用多路RPA格式来扩展RPA POC检测，以实现用于诊断数据压缩的二进制和分子编码。这项新技术依靠结合分子标记来在侧向流动膜上在空间上区分核酸种类。特别是，我们确定了可以在RPA反应过程中掺入的另外五个分子标记，以用于随后与LFD检测的偶联。与两个先前展示的成功标记相结合，我们展示了实现RPA反应的七重检测与多重LFD检测耦合的潜力。当这种七重检测与二进制和分子编码结合使用时，可以产生直观的7段输出显示。我们注意到在所有实验中，我们使用相同的DNA模板，除了正向引物上的5'标记，以消除核酸序列扩增偏倚的任何影响。

更新日期：2019-07-01

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