2.3 Rolling circle amplification

Rolling circle amplification (RCA) is another rapid and effective isothermal nucleic acid amplification method. Circular DNA template, DNA or RNA polymerase, short primer and deoxy-nucleotide triphosphates (dNTP) or nucleotide triphosphates (NTP) are the main component of the RCA reaction system (Kang et al., 2022). For RNA sequence amplification, Escherichia Coli RNA polymerase is essential for the successful running of the reaction (Lizardi et al., 1998). RCA also has the ability to integrate with other emerging technologies like nanobiotechnology and CRISPR (Kang et al., 2022), which allows it to monitor the background level and reduce the false-positive rate (Tian et al., 2020). As for SARS-CoV-2 detection, RCA-based techniques also contribute to the control of the pandemic. Liu et al. (2021) presented a completely new demonstration of the RCA-CRISPR system, which consists of a padlock probe-based RCA step and a subsequent CRISPR-Cas12a-based signal amplification step. The whole reaction takes 3 h at 30°C, with a LOD of 30.3 fM and a good specificity. Detection results of clinical samples (n = 48) by this method showed 100% concordance with RT-PCR (Liu et al., 2021). In summary, RCA is an easy and efficient isothermal enzymatic method using unique DNA and RNA polymerases with high specificity.