Near-digital amplification in paper improves sensitivity and speed in biplexed reactions.
Near-digital amplification in paper improves sensitivity and speed in biplexed reactions.
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DOI：
10.1038/s41598-022-18937-8
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发表时间：
2022-08-26
影响因子：
4.6
通讯作者：
Yager, Paul
中科院分区：
文献类型：
--
作者：
Shah, Kamal G.;Kumar, Sujatha;Yager, Paul
最简单的即时检测通常是纸和塑料设备，以低成本和最少的用户步骤检测蛋白质或核酸，尽管检测限很差。数字分析通过将样品在多个威尔斯孔（或液滴）中分开、防止扩散以及在多个小威尔斯孔中进行分析物扩增和检测来提高检测和分析物定量的极限。然而，真正的数字核酸扩增测试（NAAT）需要昂贵的耗材盒，其被精确地制造、对准和操作以实现低检测限。在这项研究中，我们演示了如何在低成本多孔介质中实现近数字NAAT，同时接近数字检测的下限。近数字NAAT是由一个含有冻干扩增试剂的纸膜实现的，该纸膜可以自动、被动地在大面积上测量和分配样本。如果在放大期间以高空间分辨率对垫成像，则在允许扩散的同时在纸膜中执行NAAT捕获了数字NAAT的许多益处。我们表明，近数字NAAT是兼容的低成本的纸和塑料一次性墨盒耦合到一个2层刚性印刷电路板加热器（MD NAAT平台）。我们还演示了与具有不同相机传感器的移动的手机的双路复用和成像的兼容性。我们表明，近数字NAAT将信噪比提高了约10倍，在含有105个拷贝的共扩增内部扩增对照DNA的双链反应中，将耐甲氧西林金黄色葡萄球菌基因组DNA的检测限从103个拷贝以上提高到100至316个拷贝，并将阳性样品的扩增时间从45分钟缩短到15-20分钟。
The simplest point-of-care assays are usually paper and plastic devices that detect proteins or nucleic acids at low cost and minimal user steps, albeit with poor limits of detection. Digital assays improve limits of detection and analyte quantification by splitting a sample across many wells (or droplets), preventing diffusion, and performing analyte amplification and detection in multiple small wells. However, truly digital nucleic acid amplification tests (NAATs) require costly consumable cartridges that are precisely manufactured, aligned, and operated to enable low detection limits. In this study, we demonstrate how to implement near-digital NAATs in low-cost porous media while approaching the low limits of detection of digital assays. The near-digital NAAT was enabled by a paper membrane containing lyophilized amplification reagents that automatically, passively meters and distributes a sample over a wide area. Performing a NAAT in the paper membrane while allowing diffusion captures many of the benefits of digital NAATs if the pad is imaged at a high spatial resolution during amplification. We show that the near-digital NAAT is compatible with a low-cost paper and plastic disposable cartridge coupled to a 2-layer rigid printed circuit board heater (the MD NAAT platform). We also demonstrate compatibility with biplexing and imaging with mobile phones with different camera sensors. We show that the near-digital NAAT increased signal-to-noise ratios by ~ 10×, improved limits of detection from above 103 copies of methicillin-resistant Staphylococcus aureus genomic DNA to between 100 and 316 copies in a biplexed reaction containing 105 copies of co-amplifying internal amplification control DNA, and reduced time-to-result from 45 min of amplification to 15–20 min for the positive samples.
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