Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription
Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription
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DOI：
10.1038/s41551-020-00617-5
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发表时间：
2020-09-18
影响因子：
28.1
通讯作者：
Lee, Jeong Wook
中科院分区：
文献类型：
--
作者：
Woo, Chang Ha;Jang, Sungho;Lee, Jeong Wook
控制病毒爆发需要灵敏、简单和快速的核酸诊断检测。在这里，我们报告了一个高度敏感和特异性的一锅法检测基于荧光的RNA从病原体。该测定可在30-50分钟的孵育时间内进行，并可达到0.1阿摩尔RNA浓度的检测限，其依赖于产生结合荧光染料的RNA适体的持续等温反应级联。RNA适体由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，所述启动子DNA探针和报告DNA探针通过SplintR连接酶（aChlorellavirus DNA连接酶）与靶单链RNA序列杂交。在40例鼻咽SARS-CoV-2样本中，该检测方法的阳性预测值和阴性预测值分别达到95%和100%。该方法可快速检测多种病毒和细菌RNA，一锅法荧光检测RNA可准确、快速地检测出特定的病毒和细菌病原体，如临床样本中的SARS-CoV-2 RNA。
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 (aChlorellavirus 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.A one-pot enzymatic assay for the fluorescence detection of RNA accurately and rapidly detects specific viral and bacterial pathogens, as shown for SARS-CoV-2 RNA in clinical samples.