Journal article
Homogeneous circle-to-circle amplification for real-time optomagnetic detection of SARS-CoV-2 RdRp coding sequence
Fluidic Array Systems and Technology, Nano and Bio-physical Systems, Department of Health Technology, Technical University of Denmark1
Magnetic Systems, Nano and Bio-physical Systems, Department of Health Technology, Technical University of Denmark2
Nano and Bio-physical Systems, Department of Health Technology, Technical University of Denmark3
Department of Health Technology, Technical University of Denmark4
Department of Physics, Technical University of Denmark5
Nanomaterials and Devices, Department of Physics, Technical University of Denmark6
BluSense Diagnostics IVS7
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 step-by-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.
Language: | English |
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Year: | 2020 |
Pages: | 112356 |
ISSN: | 18734235 and 09565663 |
Types: | Journal article |
DOI: | 10.1016/j.bios.2020.112356 |
ORCIDs: | Tian, Bo , Dufva, Martin and Hansen, Mikkel Fougt |