Department of Chemistry, University of Washington, Seattle, WA, USA.
Methods Mol Biol. 2022;2393:279-295. doi: 10.1007/978-1-0716-1803-5_15.
Digital nucleic acid quantitation methods show excellent sensitivity and specificity for pathogen detection. Droplet digital PCR (ddPCR) is the most advanced digital nucleic acid quantitation method and has been commercialized, but is not suitable for many point-of-care applications due to its complex instrumentation. Here we describe a simple microfluidics-based self-digitization (SD) chip for quantifying nucleic acids at the point of care with minimal instrumentation. We demonstrate the clinical diagnostic capability of this platform by applying it to quantifying human viral DNA and RNA. SD chips with a range of well numbers and volumes are tested, and isothermal methods are used to amplify the DNA and RNA to a detectable level. Sample concentration is determined based on the measured volume in the wells and the number of wells with fluorescence greater than a threshold based on a Poisson distribution. Concentration measurements over the low concentration range of 0-100 molecules/μL showed a strong correlation (R = 0.99) with measurements using a real-time PCR assay, demonstrating the sensitivity and specificity of the SD chip platform.
数字核酸定量方法在病原体检测方面表现出出色的灵敏度和特异性。液滴数字 PCR（ddPCR）是最先进的数字核酸定量方法，已经商业化，但由于其仪器复杂，不适合许多即时护理应用。在这里，我们描述了一种简单的基于微流控的自数字化（SD）芯片，用于在即时护理点用最小的仪器来定量核酸。我们通过应用该平台来定量检测人类病毒 DNA 和 RNA，展示了该平台的临床诊断能力。我们测试了具有一系列井数和体积的 SD 芯片，并使用等温方法将 DNA 和 RNA 扩增到可检测水平。根据孔中测量的体积和基于泊松分布的荧光强度大于阈值的孔数来确定样品浓度。在 0-100 分子/μL 的低浓度范围内的浓度测量与使用实时 PCR 测定的测量结果具有很强的相关性（R = 0.99），证明了 SD 芯片平台的灵敏度和特异性。
