Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, the Netherlands.
Amsterdam University Medical Centers, Academic Medical Centre at the University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Laboratory for Experimental Parasitology, Amsterdam institute for Infection and Immunity, Amsterdam, the Netherlands.
Nanoscale. 2022 Feb 3;14(5):1885-1895. doi: 10.1039/d1nr06557b.
Nucleic-acid detection is crucial for basic research as well as for applications in medicine such as diagnostics. In resource-limited settings, however, most DNA-detection diagnostic schemes are inapplicable since they rely on expensive machinery, electricity, and trained personnel. Here, we present an isothermal DNA detection scheme for the diagnosis of pathogenic DNA in resource-limited settings. DNA was extracted from urine and blood samples using two different instrument-free methods, and amplified using Recombinase Polymerase Amplification with a sensitivity of <10 copies of DNA within 15 minutes. Target DNA was bound by dCas9/sgRNA that was labelled with a DNA oligomer to subsequently induce Rolling Circle Amplification. This second amplification step produced many copies of a G-quadruplex DNA structure that facilitates a colorimetric readout that is visible to the naked eye. This isothermal DNA-detection scheme can be performed at temperatures between 20-45 °C. As an example of the applicability of the approach, we isothermally (23 °C) detected DNA from a parasite causing visceral leishmaniasis that was spiked into buffer and resulted in a sensitivity of at least 1 zeptomole. For proof of principle, DNA spiked into blood was coupled to the CRISPR-dCas9-based detection scheme yielding a colorimetric readout visible to the naked eye. Given the versatility of the guide-RNA programmability of targets, we envision that this DNA detection scheme can be adapted to detect any DNA with minimal means, which facilitates applications such as point-of-care diagnostics in resource-limited settings.
核酸检测对于基础研究和医学应用（如诊断）都至关重要。然而，在资源有限的环境下，大多数 DNA 检测诊断方案都不适用，因为它们依赖昂贵的仪器、电力和经过培训的人员。在这里，我们提出了一种在资源有限的环境下用于诊断致病 DNA 的等温 DNA 检测方案。使用两种不同的无仪器方法从尿液和血液样本中提取 DNA，并使用重组酶聚合酶扩增进行扩增，灵敏度在 15 分钟内达到 <10 个拷贝的 DNA。目标 DNA 被 dCas9/sgRNA 结合，该 sgRNA 用 DNA 寡核苷酸标记，随后诱导滚环扩增。这个第二扩增步骤产生了许多 G-四链体 DNA 结构的拷贝，促进了比色读出，肉眼可见。这种等温 DNA 检测方案可以在 20-45°C 之间的温度下进行。作为该方法适用性的一个例子，我们在等温（23°C）条件下检测了寄生虫引起的内脏利什曼病的 DNA，该寄生虫被添加到缓冲液中，灵敏度至少达到 1 zeptomole。为了验证原理，将血液中添加的 DNA 与基于 CRISPR-dCas9 的检测方案耦合，产生肉眼可见的比色读数。鉴于靶标引导 RNA 编程的多功能性，我们设想这种 DNA 检测方案可以通过最小的手段来检测任何 DNA，从而促进资源有限环境中的即时诊断等应用。
