Capture and detection of Staphylococcus aureus with dual labeled aptamers to cell surface components
| Autor: | N. Bhavanashri, Joseph J. Kingston, Bhairab Mondal, Padma Sudha Rani Lavu, Shylaja Ramlal |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Staphylococcus aureus Aptamer Population medicine.disease_cause 01 natural sciences Microbiology Flow cytometry 03 medical and health sciences Limit of Detection medicine Humans education Detection limit education.field_of_study medicine.diagnostic_test Base Sequence Staining and Labeling Chemistry Oligonucleotide 010401 analytical chemistry SELEX Aptamer Technique General Medicine Aptamers Nucleotide Staphylococcal Infections Flow Cytometry Molecular biology 0104 chemical sciences High-Throughput Screening Assays 030104 developmental biology Rab Systematic evolution of ligands by exponential enrichment Fluorescein-5-isothiocyanate Food Science |
| Zdroj: | International journal of food microbiology. 265 |
| ISSN: | 1879-3460 |
| Popis: | In the present study, a high throughput whole cell SELEX method has been applied successfully in selecting specific aptamers against whole cells of Staphylococcus aureus, a potent food poisoning bacterium. A total ten rounds of SELEX and three rounds of intermittent counter SELEX, was performed to obtain specific aptamers. Obtained oligonucleotide pool were cloned, sequenced and then grouped into different families based on their primary sequence homology and secondary structure similarity. FITC labeled sequences from different families were selected for further characterization via flow cytometry analysis. The dissociation constant (Kd) values of specific and higher binders ranged from 34 to 128nM. Binding assays to assess the selectivity of aptamer RAB10, RAB 20, RAB 28 and RAB 35 demonstrated high affinity against S. aureus and low binding affinity for other bacteria. To demonstrate the potential use of the aptamer a sensitive dual labeled sandwich detection system was developed using aptamer RAB10 and RAB 35 with a detection limit of 102CFU/mL. Furthermore detection from mixed cell population and spiked sample emphasized the robustness as well as applicability of the developed method. Altogether, the established assay could be a reliable detection tool for the routine investigation of Staphylococcus aureus in samples from food and clinical sources. |
| Databáze: | OpenAIRE |
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