Choi Ji Wook, Seo Won Ho, Kang Taejoon, Kang Taewook, Chung Bong Geun
Department of Mechanical Engineering, Sogang University, Seoul, Korea.
Department of Biomedical Engineering, Sogang University, Seoul, Korea.
Lab Chip. 2023 May 16;23(10):2389-2398. doi: 10.1039/d3lc00025g.
Since the outbreak of coronavirus 2019 (COVID-19), detection technologies have been attracting a great deal of attention in molecular diagnosis applications. In particular, the droplet digital PCR (ddPCR) has become a promising tool as it offers absolute quantification of target nucleic acids with high specificity and sensitivity. In recent years, the combination of the isothermal amplification strategies has made ddPCR a popular method for on-site testing by enabling amplification at a constant temperature. However, the current isothermal ddPCR assays are still challenging due to inherent non-specific amplification. In this paper, we present a multiplexed droplet digital recombinase polymerase amplification (MddRPA) with precise initiation of the reaction. First, the reaction temperature and dynamic range of reverse transcription (RT) and RPA were characterized by real-time monitoring of fluorescence intensities. Using a droplet-based microfluidic chip, the master mix and the initiator were fractionated and rapidly mixed within well-confined droplets. Due to the high heat transfer and mass transfer of the droplets, the precise initiation of the amplification was enabled and the entire assay could be conducted within 30 min. The concentrations of target RNA in the range from 5 copies per μL to 2500 copies per μL could be detected with high linearity ( > 0.999). Furthermore, the multiplexed detection of three types of human coronaviruses was successfully demonstrated with high specificity (>96%). Finally, we compared the performance of the assay with a commercial RT-qPCR system using COVID-19 clinical samples. The MddRPA assay showed a 100% concordance with the RT-qPCR results, indicating its reliability and accuracy in detecting SARS-CoV-2 nucleic acids in clinical samples. Therefore, our MddRPA assay with rapid detection, precise quantification, and multiplexing capability would be an interesting method for molecular diagnosis of viral infections.
自2019年冠状病毒病（COVID-19）爆发以来，检测技术在分子诊断应用中备受关注。特别是，液滴数字PCR（ddPCR）已成为一种有前景的工具，因为它能以高特异性和灵敏度对目标核酸进行绝对定量。近年来，等温扩增策略的结合使ddPCR成为一种通过恒温扩增实现现场检测的流行方法。然而，由于固有的非特异性扩增，目前的等温ddPCR检测仍具有挑战性。在本文中，我们提出了一种具有精确反应起始的多重液滴数字重组酶聚合酶扩增（MddRPA）方法。首先，通过实时监测荧光强度来表征逆转录（RT）和重组酶聚合酶扩增（RPA）的反应温度和动态范围。使用基于液滴的微流控芯片，将反应预混液和引发剂进行分馏，并在受限良好的液滴内快速混合。由于液滴具有高传热和传质特性，实现了扩增的精确起始，整个检测可在30分钟内完成。每微升5个拷贝至每微升2500个拷贝范围内的目标RNA浓度能够以高线性度（>0.999）进行检测。此外，成功证明了对三种人类冠状病毒的多重检测具有高特异性（>96%）。最后，我们使用COVID-19临床样本将该检测方法与商业RT-qPCR系统的性能进行了比较。MddRPA检测与RT-qPCR结果显示100%一致性，表明其在检测临床样本中SARS-CoV-2核酸方面的可靠性和准确性。因此，我们的具有快速检测、精确定量和多重检测能力的MddRPA检测方法将是一种用于病毒感染分子诊断的有趣方法。
