Department of Infectious Disease, Faculty of Medicine, Imperial College London, UK.
Department of Electrical and Electronic Engineering, Faculty of Engineering, Imperial College London, UK.
Analyst. 2023 Jun 26;148(13):3036-3044. doi: 10.1039/d3an00296a.
Nucleic acid extraction (NAE) plays a crucial role for diagnostic testing procedures. For decades, dried blood spots (DBS) have been used for serology, drug monitoring, and molecular studies. However, extracting nucleic acids from DBS remains a significant challenge, especially when attempting to implement these applications to the point-of-care (POC). To address this issue, we have developed a paper-based NAE method using cellulose filter papers (DBSFP) that operates without the need for electricity (at room temperature). Our method allows for NAE in less than 7 min, and it involves grade 3 filter paper pre-treated with 8% (v/v) igepal surfactant, 1 min washing step with 1× PBS, and 5 min incubation at room temperature in 1× TE buffer. The performance of the methodology was assessed with loop-mediated isothermal amplification (LAMP), targeting the human reference gene beta-actin and the gene from . The developed method was evaluated against FTA cards and magnetic bead-based purification, using time-to-positive (min) for comparative analysis. Furthermore, we optimised our approach to take advantage of the dual functionality of the paper-based extraction, allowing for elution () as well as direct placement of the disk in the LAMP reaction (). This flexibility extends to eukaryotic cells, bacterial cells, and viral particles. We successfully validated the method for RNA/DNA detection and demonstrated its compatibility with whole blood stored in anticoagulants. Additionally, we studied the compatibility of DBSFP with colorimetric and lateral flow detection, showcasing its potential for POC applications. Across various tested matrices, targets, and experimental conditions, our results were comparable to those obtained using gold standard methods, highlighting the versatility of our methodology. In summary, this manuscript presents a cost-effective solution for NAE from DBS, enabling molecular testing in virtually any POC setting. When combined with LAMP, our approach provides sample-to-result detection in under 35 minutes.
核酸提取（NAE）在诊断检测程序中起着至关重要的作用。几十年来，干血斑（DBS）一直用于血清学、药物监测和分子研究。然而，从 DBS 中提取核酸仍然是一个重大挑战，特别是在尝试将这些应用实施到即时护理（POC）点时。为了解决这个问题，我们开发了一种使用纤维素滤纸（DBSFP）的基于纸张的 NAE 方法，该方法无需电力（在室温下）即可运行。我们的方法允许在不到 7 分钟的时间内进行 NAE，它涉及用 8%（v/v）igepal 表面活性剂预处理的 3 级滤纸，用 1×PBS 进行 1 分钟洗涤步骤，以及在室温下 1×TE 缓冲液中孵育 5 分钟。该方法的性能通过环介导等温扩增（LAMP）进行评估，靶向人类参考基因β-肌动蛋白和. 基因。开发的方法与 FTA 卡和基于磁珠的纯化进行了比较分析，比较分析的时间为阳性时间（min）。此外，我们优化了我们的方法，以利用基于纸张的提取的双重功能，允许洗脱（）以及直接将磁盘放置在 LAMP 反应中（）。这种灵活性扩展到真核细胞、细菌细胞和病毒颗粒。我们成功地验证了该方法用于 RNA/DNA 检测，并证明了它与抗凝剂储存的全血的兼容性。此外，我们研究了 DBSFP 与比色和侧向流动检测的兼容性，展示了其在 POC 应用中的潜力。在各种测试的基质、靶标和实验条件下，我们的结果与使用金标准方法获得的结果相当，突出了我们方法的多功能性。总之，本文提出了一种从 DBS 中进行 NAE 的经济有效的解决方案，使分子测试几乎可以在任何 POC 环境中进行。当与 LAMP 结合使用时，我们的方法可以在 35 分钟内实现从样本到结果的检测。
