Selection and Characterization of Cell Surface Specific Aptamer and Development of Fluorescence Assay for Detection of Shigella flexneri from Water Samples
| Autor: | Bhairab Mondal, Padma Sudha Rani Lavu, Shylaja Ramlal |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Sociology and Political Science
Aptamer Clinical Biochemistry Biosensing Techniques 010402 general chemistry 01 natural sciences Biochemistry Flow cytometry Shigella flexneri chemistry.chemical_compound medicine Protein secondary structure Spectroscopy biology medicine.diagnostic_test 010405 organic chemistry Aptamers Nucleotide biology.organism_classification Fluorescence 0104 chemical sciences Dissociation constant Clinical Psychology Spectrometry Fluorescence chemistry Water Microbiology Law Social Sciences (miscellaneous) DNA Systematic evolution of ligands by exponential enrichment |
| Zdroj: | Journal of fluorescence. 31(3) |
| ISSN: | 1573-4994 |
| Popis: | The present study demonstrates, development of ssDNA aptamers against whole cell of S. flexneri employing a whole bacterium-based Systemic Evolution of Ligands by Exponential Enrichment (SELEX). After ten rounds of SELEX, cell surface specific aptamer pool was cloned, sequenced and divided based on sequence similarities and secondary structure. Binding affinity of FITC labelled aptamer from different group were carried out by flow cytometry analysis. The dissociation constant (Kd) values for specific and higher binder were evaluated to range from 144 to 329 nM. Six high binding aptamers with lower dissociation constant was chosen for selectivity study. Aptamer SHI 23, SHI 37 and SHI 42 showed higher selectivity towards S. flexneri in comparison with other related bacteria. Further applicability of selected aptamer was proven by fluorescence assay for convenience detection of target cell from spiked water sample and natural contaminated water samples. Altogether, aptamer generated in this study can be alternative DNA ligands for detection of S. flexneri compared to available ligands. |
| Databáze: | OpenAIRE |
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