Dual-Gene-Controlled Rolling Circle Amplification Strategy for SARS-CoV-2 Analysis.

ABSTRACT

The development of sensitive, accurate, and conveniently operated methods for the simultaneous assay of two nucleic acids is promising while still challenging. In this work, by using two genes (the N gene and RdRp gene) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as examples, we have designed an ingenious dual-gene-controlled rolling circle amplification (RCA) strategy to propose an accurate and sensitive electrochemical method. Specifically, the coexistence of the two target genes can trigger the RCA reaction to generate a number of repeated G-quadruplex (G4)-forming sequences. These sequences then switch into G4/hemin complexes with redox activity after the incubation of hemin, which can catalyze the TMB/H2O2 substrates to produce significantly enhanced current responses. Experimental results reveal that the proposed method exhibits satisfying feasibility and analytical performance, enabling the sensitive detection of SARS-CoV-2 in the range of 0.1-5000 pM, with the detection limit of 57 fM. Meanwhile, because only the simultaneous existence of the two target genes can effectively trigger the downstream amplification reaction, this method can effectively avoid false-positives and ensure specificity as well as accuracy. Furthermore, our method can distinguish the COVID-19 samples from healthy people, and the outcomes show a satisfying agreement with the results of RT-PCR, manifesting that our label-free dual-gene-controlled RCA strategy exhibits great possibility in clinical application.