Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which Nucleic Acid Sequence Based Amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon, fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform employing a cell phone camera module, compatible with field detection.

中文翻译：

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具有荧光适体读数的高度特异性、多重等温病原体检测

等温、无细胞、基于合成生物学的病原体检测方法利用生物系统中可用工具的强大功能，例如与冻干兼容的高活性聚合酶，而没有活细胞系统固有的复杂性，其中基于核酸序列的扩增(NASBA) 是众所周知的。尽管与无细胞系统相关的复杂性降低，但副反应是这些系统的共同特征。因此，这些系统通常会因缺乏扩增子的反应而出现假阳性。在这里，我们展示了包含缺乏启动子且与扩增子无关的 DNA 双链体，完全抑制了假阳性，使一组荧光适体能够用作 NASBA 标签 (Apta-NASBA)。Apta-NASBA 的检测限为 1 pM，可以提供多重、多色荧光读数。此外，Apta-NASBA 可以使用各种设备进行，例如荧光酶标仪、qPCR 仪器或采用手机摄像头模块的超低成本 Raspberry Pi 3D 打印检测平台，与场检测。