Abstract
Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX) is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, selected, and developed two categories of aptamers that were selected by two kinds of whole-cell SELEX, by mixing and combining FACS analysis and a counter-SELEX process. Using this approach, we have developed a biosensor system that employs a high affinity aptamer for detection of target bacteria. FAM-labeled aptamer sequences with high binding to S. aureus, as determined by fluorescence spectroscopic analysis, were identified, and aptamer A14, selected by the basic whole-cell SELEX using a once-off FACS analysis, and which had a high binding affinity and specificity, was chosen. The binding assay was evaluated using FACS analysis. Our study demonstrated the development of a set of whole-cell SELEX derived aptamers specific to S. aureus; this approach can be used in the identification of other bacteria.
Highlights
Enterotoxin-producing Staphylococcus aureus (S. aureus) cause nausea, stomach cramps, vomiting, and diarrhea [1]
After incubation with non-target bacteria, bound ssDNA was removed by centrifugation, while the unbound aptamer candidates in the supernatant were collected for one more round of counter-Systemic Evolution of Ligands by Exponential enrichment (SELEX) or SELEX
The mean fluorescence intensity of target bacteria labeled with aptamer candidates was used to calculate the specific binding by subtracting the mean fluorescence intensity of nonspecific binding produced by unselected library DNA
Summary
Enterotoxin-producing Staphylococcus aureus (S. aureus) cause nausea, stomach cramps, vomiting, and diarrhea [1]. Methods for detection and control of S. aureus are well established, conventional methods, such as the standard plate count method after selective enrichment in broth, are lengthy, complicated and require multiple steps [4,7] To overcome these factors, biosensors that allow rapid and efficient detection of foodborne bacteria have been developed [8,9]. Because flow cytometry is a crucial method for selecting target aptamers bound to cells [20], multiple FACS sorting steps were incorporated in the modified SELEX process to further improve the specificity. We compared their binding activity using FACS analysis
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