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    Multiple detection method for SARS-CoV-2 with aptamer cocktails depending on their localization in paper-based assays
    (Bmc, 2025) Cam Derin, Dilek; Gunduz, Elif
    BackgroundThe nucleocapsid (N) and spike (S) proteins are key markers for SARS-CoV-2 diagnosis and treatment because of their antigenic regions. Aptamers targeting these proteins have been developed for various diagnostic platforms, including Lateral Flow Assays (LFAs). However, existing studies have focused mainly on the use of single aptamer or antibody/aptamer sandwiches in capture zones, lacking the advantages of using aptamer cocktails. The present study aimed to develop multiple aptamer cocktail-based lateral flow assays (mACLFAs) for rapid and accurate detection of SARS-CoV-2 in clinical samples.MethodsSpherical gold nanoparticles were synthesized and used as labels for aptamer conjugation for naked-eye analysis. Aptamers specific to the S and N proteins of SARS-CoV-2 were chosen and used as either capture or detection reagents for the sandwich assay model. A nitrocellulose membrane was used to develop the mACLFAs.ResultsModel 2 was the most efficient multiple-assay model for viral diagnosis, although models 1 and 3 correctly detected the virus in clinical samples. Model 4 and Model 5 had nonspecific interactions and were not preferred for multiple assay development. The specificity of models 1, 2 and 3 was 100%, and the sensitivity of models 2 and 3 was 100%, while it was 96.6% for Model 1.ConclusionOur findings demonstrated that the accuracy of viral detection was improved by the use of aptamer cocktails, as both the N and S proteins were captured in clinical samples. The results also highlighted the importance of the position of the detector aptamers in the capture zones in terms of the accuracy and interaction of the aptamers in the sandwich assay. Thus, these findings will be valuable for developing diagnostic tools to monitor and prevent future outbreaks, such as monkey pox virus.
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    Single-Chain Variable Fragment-Based Dot Blot, Single, and Multiple Assays for Rapid SARS-CoV-2 Diagnostics
    (Oxford Univ Press Inc, 2025) Cam Derin, Dilek; Gultekin, Enes; Taskin, Irmak Icen; Dundar, Muhammed; Otlu, Baris
    Background Antigenic detection is reliably utilized in rapid diagnostic tests and provides a significant time advantage during pandemics and epidemics. Therefore, the rapid detection of viral infections is of great importance and will remain crucial in the future. The SARS-CoV-2 outbreak, which resulted in severe losses, is the most recent example of this necessity. Among rapid diagnostic tests, lateral flow assays (LFAs) are the most practical and do not require specialized equipment, typically being developed using antibody pairs.Objective This study aimed to recombinantly produce a single-chain variable fragment (scFv) specific to the SARS-CoV-2 spike receptor-binding domain (sRBD) and to employ it in the development of LFAs utilizing both antibody and aptamer pairs and an aptamer cocktail.Methods Gold nanoparticles were employed as labeling agents, while both the scFv and full length forms of CR3022, along with aptamers specific to the S and N proteins, were utilized in a sandwich assay format.Results scFv was produced at a higher concentration and biologically active. It demonstrated effective viral detection in single LFA, dot blot assay (DBA), and multiplex LFA. While single LFA successfully detected only the synthetic target, DBA and multiplex LFA selectively identified the virus in nasopharyngeal and oropharyngeal swab samples.Conclusion Findings highlight the differences and effectiveness of using scFv in combination with other capture agents and different assay principles for the development of cost-effective and rapid diagnostic tests.Highlights scFvs exhibit variable binding in sandwich assays depending on the combinations employed. When used in combination with an aptamer cocktail, scFvs demonstrate enhanced target binding, which is shown for the first time in this study. The use of multiple testing strategies enables a more effective viral diagnosis.