Aptamer Sandwich Lateral Flow Assay (AptaFlow) for Antibody-Free SARS-CoV-2 Detection

Area of Science:

• Biotechnology
• Molecular Biology
• Infectious Disease Diagnostics

Background:

• The COVID-19 pandemic necessitates rapid diagnostic tools to limit viral transmission.
• Existing tests require improvement in speed and cost-effectiveness.
• DNA aptamers offer a promising alternative for sensitive and specific pathogen detection.

Purpose of the Study:

• To identify and characterize novel DNA aptamers targeting the SARS-CoV-2 spike protein N-terminal domain (NTD).
• To develop a rapid, low-cost lateral flow assay (LFA) for SARS-CoV-2 detection using aptamers.
• To evaluate the performance of the aptamer-based assay for detecting infectious SARS-CoV-2 virus.

Main Methods:

• Selection and characterization of DNA aptamers binding to the SARS-CoV-2 spike protein NTD.
• Development of an aptamer sandwich lateral flow assay (AptaFlow) utilizing two aptamers (SNAP1 and SNAP4).
• Quantification of SARS-CoV-2 detection limits using UV-inactivated virus in the AptaFlow assay.

Main Results:

• A new high-affinity DNA aptamer, SNAP4, was identified, exhibiting a 2-fold improvement in binding affinity (<30 nM) over the previously identified SNAP1.
• The AptaFlow assay, incorporating both SNAP1 and SNAP4, successfully detected SARS-CoV-2 UV-inactivated virus at concentrations as low as 10^6 copies/mL.
• AptaFlow demonstrated a production cost of less than $1 per test and provided results within one hour.

Conclusions:

• The AptaFlow assay, utilizing SNAP1 and SNAP4 aptamers, provides a sensitive and rapid method for detecting SARS-CoV-2.
• This aptamer-based LFA is a cost-effective and convenient antigen test suitable for controlling the COVID-19 pandemic.
• The developed assay has the potential to detect individuals with high viral loads, indicating a high probability of transmission.