Abstract:
The exponential spread of COVID-19 has prompted the need to develop
a simple and sensitive diagnostic tool. Aptamer-based detection assays like
ELONA are promising since they are inexpensive and sensitive. Aptamers have
advantages over antibodies in wide modification, small size, in vitro selection,
and stability under stringent conditions, which aid in scalable and reliable
detection. In this work, we used aptamers against SARS-CoV-2 RBD S protein
to design a simple and sensitive ELONA detection tool. Screening CoV2-RBD-1C
and CoV2-RBD-4C aptamers and optimizing assay conditions led to the
development of a direct ELONA that can detect SARS-CoV-2 RBD S
glycoprotein in buffer solution and 0.1 % human nasal fluid with a detection
limit of 2.16 ng/mL and 1.02 ng/mL, respectively. We detected inactivated Alpha,
Wuhan, and Delta variants of SARS-CoV-2 with the detection limit of 3.73, 5.72,
and 6.02 TCID50/mL, respectively. Using the two aptamers as capture and
reporter elements, we designed a more sensitive sandwich assay to identify
the three SARS-CoV-2 variants employed in this research. As predicted, a lower
detection limit was obtained. Sandwich assay LOD was 2.31 TCID50/mL for
Alpha, 1.15 TCID50/mL for Wuhan, and 2.96 TCID50/mL for Delta. The sensitivity
of sandwich ELONA was validated using Alpha and Wuhan variants spiked in 0.1%
human nasal fluid sample condition and were detected in 1.41 and 1.79 TCID50/
mL LOD, respectively. SEM was used to visualize the presence of viral particles in
the Delta variant sample. The effective detection of SARS-CoV-2 in this study
confirms the potential of our aptamer-based technique as a screening tool.