Digital Quantification of Human Viral RNA and DNA Using a Self-Digitization Chip.
Digital Quantification of Human Viral RNA and DNA Using a Self-Digitization Chip.
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使用自数字化芯片对人类病毒 RNA 和 DNA 进行数字定量。
DOI：
10.1007/978-1-0716-1803-5_15
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Chiu,DanielT
中科院分区：
文献类型：
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作者：
Wang,Jiasi;Kreutz,JasonE;Chiu,DanielT
数字核酸定量方法显示出优异的灵敏度和特异性用于病原体检测。液滴数字PCR（ddPCR）是最先进的数字核酸定量方法，并且已经商业化，但是由于其复杂的仪器而不适合于许多护理点应用。在这里，我们描述了一种简单的基于微流体的自数字化（SD）芯片，用于在护理点用最少的仪器定量核酸。我们通过将其应用于定量人类病毒DNA和RNA来证明该平台的临床诊断能力。测试具有一系列孔数和体积的SD芯片，并使用等温方法将DNA和RNA扩增至可检测水平。基于所测量的威尔斯孔中的体积和荧光大于基于泊松分布的阈值的威尔斯孔的数量来确定样品浓度。在0-100个分子/μL的低浓度范围内的浓度测量结果显示与使用实时PCR测定的测量结果具有强相关性（R2= 0.99），证明了SD芯片平台的灵敏度和特异性。
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 (R2= 0.99) with measurements using a real-time PCR assay, demonstrating the sensitivity and specificity of the SD chip platform.