Simple, Visual, Point-of-Care SARS-CoV-2 Detection Incorporating Recombinase Polymerase Amplification and Target DNA-Protein Crosslinking Enhanced Chemiluminescence

The ongoing COVID-19 pandemic, driven by persistent SARS-CoV-2 transmission, threatens human health worldwide, underscoring the urgent need for an efficient, low-cost, rapid SARS-CoV-2 detection method. Herein, we developed a point-of-care SARS-CoV-2 detection method incorporating recombinase polymerase amplification (RPA) and DNA-protein crosslinking chemiluminescence (DPCL) (RPADPCL). RPADPCL involves the crosslinking of biotinylated double-stranded RPA DNA products with horseradish peroxidase (HRP)-labeled streptavidin (SA-HRP). Modified products are captured using SA-labeled magnetic beads, and then analyzed using a chemiluminescence detector and smartphone after the addition of a chemiluminescent substrate. Under optimal conditions, the RPADPCL limit of detection (LOD) was observed to be 6 copies (within the linear detection range of 1-300 copies) for a plasmid containing the SARS-CoV-2 N gene and 15 copies (within the linear range of 10-500 copies) for in vitro transcribed (IVT) SARS-CoV-2 RNA. The proposed method is convenient, specific, visually intuitive, easy to use, and does not require external excitation. The effective RPADPCL detection of SARS-CoV-2 in complex matrix systems was verified by testing simulated clinical samples containing 10% human saliva or a virus transfer medium (VTM) spiked with a plasmid containing a SARS-CoV-2 N gene sequence or SARS-CoV-2 IVT RNA. Consequently, this method has great potential for detecting targets in clinical samples.