Fluorescence-Linked Aptamer Assay for SARS-CoV-2 Spike-Protein: A Step-by-Step Performance Analysis in Clinical...

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Infectious Disease Research

Background:

• The COVID-19 pandemic necessitates affordable and reliable diagnostic tools for widespread monitoring.
• Previous isolation of specific DNA aptamers (C7/C9) targeting the SARS-CoV-2 Spike protein required clinical validation.

Purpose of the Study:

• To assess the performance of a novel fluorescence-linked aptamer assay (FLAA) using C7/C9 aptamers in clinical samples.
• To identify optimal conditions and influential factors for the C7/C9 FLAA test.

Main Methods:

• Collected 242 clinical samples using three different methods.
• Performed fluorescence-linked aptamer assays (FLAA) with C7/C9 aptamers and two readout protocols.
• Employed statistical analyses, including agreement tests and logistic regressions, to evaluate test performance and identify influential factors.

Main Results:

• RTqPCR threshold cycles, symptom onset, and processing time significantly influenced FLAA test outcomes.
• Naturally occurring mutations, specifically D614G and T732A in the Spike protein, were detected and analyzed for their impact on aptamer recognition.
• The study established a foundational methodology for evaluating aptamer-based diagnostic tests in diverse clinical settings.

Conclusions:

• The C7/C9 aptamer-based FLAA demonstrates potential as a diagnostic tool for SARS-CoV-2.
• Further research is needed to fully elucidate the role of specific mutations in aptamer binding and to refine the diagnostic assay.
• This work provides a framework for developing and validating new aptamer-based diagnostic tests.