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A framework for the development and validation of an aptamer-based assay for pathogen detection using SARS-CoV-2 as a
Steev Loyola1,2,3, Leonardo J Monroy-Cruz4,5, Miguel Quiliano5
1Unidad de Investigación Molecular (UNIMOL), Facultad de Medicina, Universidad de Cartagena, Cartagena, Colombia. steev.loyola@gmail.com.
Scientific Reports
|May 28, 2026
Summary
This study presents a practical framework for developing aptamer-based diagnostic tests, crucial for low-resource settings. The developed aptamer-based qPCR assay showed high accuracy for detecting SARS-CoV-2 Omicron variants.
Area of Science:
- Biotechnology
- Molecular Diagnostics
- Infectious Disease Research
Background:
- COVID-19 pandemic highlighted global diagnostic disparities, especially in low- and middle-income countries (LMICs).
- Existing diagnostic methods and reliance on external technologies limit rapid response capabilities in LMICs.
- Aptamers offer a promising alternative for diagnostics, but validated frameworks for their development and application in LMICs are scarce.
Purpose of the Study:
- To present an integrated, stepwise framework for aptamer-based diagnostic tool development and validation.
- To demonstrate the feasibility of this framework using SARS-CoV-2 as a model pathogen.
- To support enhanced diagnostic preparedness and response capacity in resource-limited settings.
Main Methods:
- Systematic Evolution of Ligands by Exponential Enrichment (SELEX) protocol adapted for low- to medium-complexity laboratories.
- High-throughput sequencing and bioinformatic analysis to identify candidate aptamer sequences.
- Development and validation of a DNA aptamer-based quantitative PCR (Apta-qPCR) assay using clinical samples.
Main Results:
- Aptamer APT-35b was identified and incorporated into the Apta-qPCR assay.
- The Apta-qPCR assay demonstrated 80.8% sensitivity and 95.3% specificity for SARS-CoV-2 Omicron BA.1/BA.1.1.
- No cross-reactivity was observed with other human coronaviruses, though performance varied with more evolved Omicron subvariants.
Conclusions:
- The developed framework provides a practical roadmap linking aptamer discovery to functional assay development.
- The Apta-qPCR assay shows potential for diagnosing SARS-CoV-2 variants, particularly in LMICs.
- This adaptable framework can be applied to diverse pathogens, enhancing global diagnostic capabilities.
