Dumbbell-type triplex molecular switch-based logic molecular assays of SARS-CoV-2

Accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of great importance to control the COVID-19 pandemic. The gold standard assays for COVID-19 diagnostics are mainly based on separately detecting open reading frame 1ab (ORF1ab) and nucleoprotein (N) genes by RT-PCR. However, the current approaches often obtain false positive-misdiagnose caused by cross-contamination or undesired amplification. To address this issue, herein, we proposed a dumbbell-type triplex molecular switch (DTMS)-based, logic-gated strategy for high-fidelity SARS-CoV-2 RNA detection. The DTMS consists of a triple-helical stem region and two-loop regions for recognizing the ORF1ab and N genes of SARS-CoV-2. Only when the ORF1ab and N gene are concurrent, DTMS experiences a structural rearrangement, thus, bringing the two pyrenes into spacer proximity and leading to a new signal readout. This strategy allows detecting SARS-CoV-2 RNA with a detection limit of 1.3 nM, independent of nucleic acid amplification, holding great potential as an indicator probe for screening of COVID-19 and other population-wide epidemics.