A variety of automated sample-in-answer-out systems for in vitro molecular diagnostics have been presented and even commercialized. Although efficient in operation, they are incapable of quantifying targets, since quantitation based on analog analytical methods (via standard curve analysis) is complex, expensive, and challenging. To address this issue, herein, we describe an integrated sample-in-digital-answer-out (SIDAO) diagnostic system incorporating DNA extraction and digital recombinase polymerase amplification, which enables rapid and quantitative nucleic acid analysis from bodily fluids within a disposable cartridge. Inside the cartridge, reagents are pre-stored in sterilized tubes, with an automated pipetting module allowing facile liquid transfer. For digital analysis, we fabricate a simple, single-layer polydimethylsiloxane microfluidic device and develop a novel and simple sample compartmentalization strategy. Sample solution is partitioned into an array of 40,044 fL-volume microwells by sealing the microfluidic device through the application of mechanical pressure. The entire analysis is performed in a portable, fully automated instrument. We evaluate the quantitative capabilities of the system by analyzing Mycobacterium tuberculosis genomic DNA from both spiked saliva and serum samples, and demonstrate excellent analytical accuracy and specificity. This SIDAO system provides a promising diagnostic platform for quantitative nucleic acid testing at the point-of-care.

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已经提出了多种用于体外分子诊断的自动进样系统，甚至已经商业化。尽管操作效率很高，但是它们无法量化目标，因为基于模拟分析方法（通过标准曲线分析）的量化是复杂，昂贵且具有挑战性的。为了解决这个问题，在这里，我们描述了一个集成的数字输入/输出应答（SIDAO）诊断系统，该系统结合了DNA提取和数字重组酶聚合酶扩增功能，可以对一次性盒中的体液进行快速而定量的核酸分析。试剂盒内部将试剂预先存储在灭菌管中，并带有自动移液模块，可轻松进行液体转移。为了进行数字分析，我们制作了一个简单的，单层聚二甲基硅氧烷微流体装置，并开发了一种新颖而简单的样品区室化策略。通过施加机械压力密封微流体装置，将样品溶液划分为40,044 fL体积的微孔阵列。整个分析在便携式的全自动仪器中进行。我们通过分析评估系统的定量能力唾液和血清样品中的结核分枝杆菌基因组DNA均具有出色的分析准确性和特异性。该SIDAO系统为现场即时核酸定量检测提供了一个有前途的诊断平台。

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