School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Republic of Korea.
Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Republic of Korea.
Nat Biomed Eng. 2020 Dec;4(12):1168-1179. doi: 10.1038/s41551-020-00617-5. Epub 2020 Sep 18.
The control of viral outbreaks requires nucleic acid diagnostic tests that are sensitive, simple and fast. Here, we report a highly sensitive and specific one-pot assay for the fluorescence-based detection of RNA from pathogens. The assay, which can be performed within 30-50 min of incubation time and can reach a limit of detection of 0.1-attomolar RNA concentration, relies on a sustained isothermal reaction cascade producing an RNA aptamer that binds to a fluorogenic dye. The RNA aptamer is transcribed by the T7 RNA polymerase from the ligation product of a promoter DNA probe and a reporter DNA probe that hybridize with the target single-stranded RNA sequence via the SplintR ligase (a Chlorella virus DNA ligase). In 40 nasopharyngeal SARS-CoV-2 samples, the assay reached positive and negative predictive values of 95 and 100%, respectively. We also show that the assay can rapidly detect a range of viral and bacterial RNAs.
病毒爆发的控制需要敏感、简单和快速的核酸诊断测试。在这里，我们报告了一种基于荧光的用于检测病原体 RNA 的高度敏感和特异的一步法检测。该检测可在孵育时间 30-50 分钟内进行，检测限可达 0.1 皮摩尔 RNA 浓度，依赖于持续的等温反应级联，产生与荧光染料结合的 RNA 适体。RNA 适体由 T7 RNA 聚合酶转录，来自启动子 DNA 探针和报告 DNA 探针的连接产物，通过 SplintR 连接酶（一种衣藻病毒 DNA 连接酶）与靶单链 RNA 序列杂交。在 40 个鼻咽 SARS-CoV-2 样本中，该检测的阳性和阴性预测值分别为 95%和 100%。我们还表明，该检测可以快速检测一系列病毒和细菌 RNA。
