Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription

Abstract

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.

CRISPR/Cas9-Based Lateral Flow and Fluorescence Diagnostics

“…To further define the conditions for field-based testing, we explored the optimal settings for rapid amplification and detection via LFA. Previous studies using Cas enzymes have employed LAMP PCR and Cas in a ‘one pot’ approach for simultaneous amplification and detection [ 8 , 45 ]. In our system, this strategy yielded high levels of false positives even in the presence of large amounts of competitor soak DNA ( Figure 3 A).…”

Detection of Coronaviruses Using RNA Toehold Switch Sensors

“…Currently, the world faces unprecedented challenges by COVID-19 caused by SARS-CoV-2 and this fast-spreading pandemic disease is threatening global health. To prevent the spread of such viruses, a rapid and simple point-of-care (POC) diagnosis is needed [ 2 , 3 ].…”

Diagnostyka zakażeń SARS-CoV-2

“…Według obowiązujących w Polsce rekomendacji, szybkie testy molekularne z zastosowaniem technologii RT-PCR i izotermicznej, wykrywające RNA wirusa, mogą być wykonywane w przypadkach pilnych. Czas otrzymania wyniku w przypadku szybkich testów kasetkowych wynosi zwykle około 15-45 minut [11,15]. Przykładem takiego testu jest CovidNudge, wprowadzony w maju 2020 w szpitalach brytyjskich.…”