Abstract:
:Alternative ligands such as nucleic acid aptamers can be used for pathogen capture and detection and offer advantages over antibodies, including reduced cost, ease of production and modification, and improved stability. DNA aptamers demonstrating binding specificity to Salmonella enterica serovar Typhimurium were identified by whole-cell-systematic evolution of ligands by exponential enrichment (SELEX) beginning with a combinatorial library of biotin-labeled single stranded DNA molecules. Aptamer specificity was achieved using whole-cell counter-SELEX against select non-Salmonella genera. Aptamers binding to Salmonella were sorted, cloned, sequenced, and characterized for binding efficiency. Out of 18 candidate aptamers screened, aptamer S8-7 showed relatively high binding affinity with an apparent dissociation constant (K d value) of 1.73 ± 0.54 μM and was selected for further characterization. Binding exclusivity analysis of S8-7 showed low apparent cross-reactivity with other foodborne bacteria including Escherichia coli O157: H7 and Citrobacter braakii and moderate cross-reactivity with Bacillus cereus. Aptamer S8-7 was successfully used as a ligand for magnetic capture of serially diluted Salmonella Typhimurium cells, followed by downstream detection using qPCR. The lower limit of detection of the aptamer magnetic capture-qPCR assay was 10(2)-10(3) CFU equivalents of Salmonella Typhimurium in a 290-μl sample volume. Mean capture efficiency ranged from 3.6 to 12.6 %. Unique aspects of the study included (a) the use of SELEX targeting whole cells; (b) the application of flow cytometry for aptamer pool selection, thereby favoring purification of ligands with both high binding affinity and targeting abundant cell surface moieties; and (c) the use of pre-labeled primers that circumvented the need for post-selection ligand labeling. Taken together, this study provides proof-of-concept that biotinylated aptamers selected by whole-cell SELEX can be used in a qPCR-based capture-detection platform for Salmonella Typhimurium.
journal_name
Appl Microbiol Biotechnoljournal_title
Applied microbiology and biotechnologyauthors
Dwivedi HP,Smiley RD,Jaykus LAdoi
10.1007/s00253-013-4766-4subject
Has Abstractpub_date
2013-04-01 00:00:00pages
3677-86issue
8eissn
0175-7598issn
1432-0614journal_volume
97pub_type
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