Simultaneous Detection of SARS-CoV-2 Nucleocapsid Protein and RNA by Aptamer-Based Proximity Ligation and Quantitative PCR.

The COVID-19 pandemic underscored the global need for rapid, sensitive, and multiplexed diagnostic assays for viral detection. Real-time quantitative polymerase chain reaction (RT-qPCR) remains the standard for SARS-CoV-2 RNA detection, while antigen-based protein assays provide faster, though less sensitive, alternatives. Here, we present a novel diagnostic platform that combines proximity ligation of aptamers (PLA) with RT-qPCR to enable simultaneous detection of both SARS-CoV-2 RNA and nucleocapsid (N) protein in a single vial. Six high-affinity aptamers against the N protein were identified via capillary electrophoresis-based systematic evolution of ligands by exponential enrichment (CE-SELEX). From these, ECK1 and ECK4 were selected based on binding affinity and spatial compatibility for PLA. The aptamer pair enabled target-induced ligation followed by detection using Cy5-labeled TaqMan probes. Concurrently, SARS-CoV-2 RNA was detected with FAM-labeled probes in the same RT-qPCR reaction. This dual-analyte assay was evaluated in buffer and complex biological matrices such as saliva. Sensitivity was further enhanced by integrating droplet digital PCR (ddPCR). Aptamer binding sites at the N protein were identified via diethylpyrocarbonate (DEPC) labeling and bottom-up proteomics. Our method introduces a scalable and adaptable strategy for multiplexed pathogen diagnostics with minimal sample processing.