Although assays based on quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR) have been extremely successful for detecting SARS-CoV-2, the high cost and slow turnaround time prevent its use as a rapid screening tool for the general public ( 2).

The strategy of large-scale testing, contact tracing and isolation has been implemented by countries all over the world, which has been proven effective in controlling the spread of SARS-CoV-2. The COVID-19 pandemic, caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), has resulted in enormous loss of human life and economic burden to our society ( 1). A colorimetric sandwich assay was developed using MSA1 as both the recognition element and detection element, which was capable of detecting the pseudotyped lentivirus in 50% saliva with a limit of detection of 400 fM, confirming the potential of these aptamers as diagnostic tools for COVID-19 detection. Secondary structure prediction and sequence truncation experiments revealed that both MSA1 and MSA5 adopted a hairpin structure, which was the motif pre-designed into the original library. MSA1 and MSA5 also recognized the pseudotyped lentivirus of SARS-CoV-2 with respective K d values of 22.7 pM and 11.8 pM. Two aptamers, named MSA1 ( K d = 1.8 nM) and MSA5 ( K d = 2.7 nM), were assessed for binding to the heat-treated S1 protein, untreated S1 protein spiked into 50% human saliva and the trimeric spike protein of both the wildtype and the B.1.1.7 variant, demonstrating comparable affinities in all cases. The top 10 sequences all exhibited strong binding to the S1 protein. Using a pool of pre-structured random DNA sequences, we obtained over 100 candidate aptamers after 13 cycles of enrichment under progressively more stringent selection pressure. We performed in vitro selection experiments to identify DNA aptamers for the S1 subunit of the SARS-CoV-2 spike protein (S1 protein).

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