Rolling circle amplification with electrochemical detection assay for SARS-CoV-2 

Abstract This protocol describes the rolling circle amplification (RCA) and electrochemical detection of SARS-CoV-2. The procedure consists of 3 parts, which are the amplification, hybridization and detection steps. In the presence of RNA template, the circular DNA template will be ligated to produce a Padlock DNA, which serves as a template for amplification by phi29 DNA polymerase to produce RCA amplicons. The RCA amplicon is a concatemer containing multiple repeats of sequences that are complementary to the circular template. The RCA amplicons are hybridized with redox active probes and detected by electrochemical biosensor using differential pulse voltammetry (DPV). Due to its isothermal nature, RCA can be performed using a simple water bath or heating block. Overall, the whole assay takes approximately 45 min. The assay enables rapid, quantitative results to be obtained for detection of SARS-CoV-2, either in the laboratory or more importantly, in a field setting.