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.
The recent outbreaks of Ebola, Zika, MERS, and SARS-CoV-2 (2019-nCoV) require fast, simple, and sensitive onsite nucleic acid diagnostics that can be developed rapidly to prevent the spread of diseases. We have developed a SENsitive Splint-based one-step isothermal RNA detection (SENSR) method for rapid and straightforward onsite detection of pathogen RNAs with high sensitivity and specificity. SENSR consists of two simple enzymatic reactions: a ligation reaction by SplintR ligase and subsequent transcription by T7 RNA polymerase. The resulting transcript forms an RNA aptamer that induces fluorescence. Here, we demonstrate that SENSR is an effective and highly sensitive method for the detection of the current epidemic pathogen, severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). We also show that the platform can be extended to the detection of five other pathogens. Overall, SENSR is a molecular diagnostic method that can be developed rapidly for onsite uses requiring high sensitivity, specificity, and short assaying times.
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