Homogeneous circle-to-circle amplification for real-time optomagnetic detection of SARS-CoV-2 RdRp coding sequence

Abstract: Circle-to-circle amplification (C2CA) is a specific and precise cascade nucleic acid amplification method consisting of more than one round of padlock probe ligation and rolling circle amplification (RCA). Although C2CA provides a high amplification efficiency with a negligible increase of false-positive risk, it contains several stepby-step operation processes. We herein demonstrate a homogeneous and isothermal nucleic acid quantification strategy based on C2CA and optomagnetic analysis of magnetic nanoparticle (MNP) assembly. The proposed
homogeneous circle-to-circle amplification eliminates the need for additional monomerization and ligation steps
after the first round of RCA, and combines two amplification rounds in a one-pot reaction. The second round of
RCA produces amplicon coils that anneal to detection probes grafted onto MNPs, resulting in MNP assembly that
can be detected in real-time using an optomagnetic sensor. The proposed methodology was applied for the
detection of a synthetic complementary DNA of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2,
also known as 2019-nCoV) RdRp (RNA-dependent RNA polymerase) coding sequence, achieving a detection limit
of 0.4 fM with a dynamic detection range of 3 orders of magnitude and a total assay time of ca. 100 min. A
mathematical model was set up and validated to predict the assay performance. Moreover, the proposed method
was specific to distinguish SARS-CoV and SARS-CoV-2 sequences with high similarity.

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