A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a
A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a
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DOI：
10.1371/journal.pone.0220091
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发表时间：
2019-12-18
期刊：
影响因子：
3.7
通讯作者：
Heymann, Michael
中科院分区：
文献类型：
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作者：
Katzmeier, Florian;Aufinger, Lukas;Heymann, Michael
低资源环境下的护理点检测(POCT)需要能够独立于典型实验室基础设施运行的工具。由于其良好的信号背景比，各种各样的生物医学测试使用荧光作为读数。然而，荧光技术通常需要昂贵或复杂的仪器，很难适用于POCT。为了解决这个问题，我们开发了一种袖珍尺寸的荧光探测器，成本不到15美元，易于制造，可以在低资源环境下操作。它由标准电子元件组成，包括LED和光敏电阻、滤光片和3D打印部件，并可靠地达到检测下限(LOD)。6.8 NM荧光素，足以遵循POCT应用中使用的典型生化反应。所有的分析都是在滤纸上进行的，这允许使用扁平的探测器架构来改进信号采集。我们通过量化体外RNA转录来验证该设备，并使用基于Cas13a的荧光分析方法演示了对目标RNA的序列特异性检测，LOD为3.7 nM。Cas13a是一种RNA导向、RNA靶向的CRISPR效应器，在识别其RNA靶标后具有混杂的RNase活性。Cas13a传感具有高度的特异性和适应性，与我们的检测器相结合，是一种有希望的核酸POCT方法。此外，我们的开源设备可以用于教育环境，通过为定量分析提供低成本的仪器，或者作为未来整合硬件、软件和生物化学概念的平台。
Point-of-care testing (POCT) in low-resource settings requires tools that can operate independently of typical laboratory infrastructure. Due to its favorable signal-to-background ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, fluorescence techniques often require expensive or complex instrumentation and can be difficult to adapt for POCT. To address this issue, we developed a pocket-sized fluorescence detector costing less than $15 that is easy to manufacture and can operate in low-resource settings. It is built from standard electronic components, including an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably reaches a lower limit of detection (LOD) of. 6.8 nM fluorescein, which is sufficient to follow typical biochemical reactions used in POCT applications. All assays are conducted on filter paper, which allows for a flat detector architecture to improve signal collection. We validate the device by quantifying in vitro RNA transcription and also demonstrate sequence-specific detection of target RNAs with an LOD of 3.7 nM using a Cas13a-based fluorescence assay. Cas13a is an RNA-guided, RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of its RNA target. Cas13a sensing is highly specific and adaptable and in combination with our detector represents a promising approach for nucleic acid POCT. Furthermore, our open-source device may be used in educational settings, through providing low cost instrumentation for quantitative assays or as a platform to integrate hardware, software and biochemistry concepts in the future.