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.

控制病毒爆发需要灵敏、简单、快速的核酸诊断检测。在这里，我们报告了一种高度灵敏和特异性的一锅法，用于基于荧光检测病原体 RNA。该测定可在 30-50 分钟的孵育时间内进行，可达到 0.1 摩尔 RNA 浓度的检测极限，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 适配体由 T7 RNA 聚合酶从启动子 DNA 探针和报告 DNA 探针的连接产物转录，通过 SplintR 连接酶（一种小球藻）与靶单链 RNA 序列杂交病毒 DNA 连接酶）。在 40 份鼻咽 SARS-CoV-2 样本中，该测定分别达到了 95% 和 100% 的阳性和阴性预测值。我们还表明，该测定可以快速检测一系列病毒和细菌 RNA。