Selection of DNA aptamers for capture and detection of Salmonella Typhimurium using a whole-cell SELEX approach in conjunction with cell sorting

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.

Selection of Peptidoglycan-Specific Aptamers for Bacterial Cells Identification

“…The anti-peptidoglycan aptamers selected in the present work were able to distinguish bacteria from fungi cells in vitro and also showed low binding to representative human cells, fulfilling the requirement to act as potential probes for the development of bacterial infection diagnostic radiopharmaceuticals. Aptamers have been previously selected against purified bacterial components, like bacterial toxins [19], purified antigens [20], outer membrane proteins [21], and whole cells, including Mycobacterium tuberculosis [22], S. aureus [23], Streptococcus pyogenes [24], and Salmonella Typhimurium [25]. Aptamers against autoclaved anthrax spores were also reported [26].…”

Identification and application of ssDNA aptamers against H37Rv in the detection of Mycobacterium tuberculosis

“…Since the entirety of the cell surface targets in their native three-dimensional conformations will be available in these SELEX processes (Dwivedi et al 2013;Shamah et al 2008), aptamers derived in this way would be a greater success in targeting pathogen cells than those generated from purified targets (Guo et al 2008). Currently, many aptamers targeting pathogen cells have been developed by the whole cell SELEX and used as the capture or detection molecules in the detection of pathogen (Dwivedi et al 2010;Yang et al 2013).…”

“…Bacteriological detection based on aptamer has been reported in many pathogenic bacteria such as Salmonella typhimurium, Pseudomonas aeruginosa, and Staphylococcus aurous and showed high specificity in the discrimination of these pathogenic bacteria (Dwivedi et al 2013;Han and Lee 2013;Wang et al 2011).…”

An Update on Aptamer-Based Multiplex System Approaches for the Detection of Common Foodborne Pathogens

“…In this study, primers were prelabeled and obtained DNA aptamer pool has been sorted using FACS followed by biotinylation. It displayed less cross-reactivity to other foodborne pathogens and possessed good Kd of 1.73 ± 0.54 μM detected in low sample of 290 μL containing 10 2 -10 3 cfu/mL (Dwivedi et al 2013). The use of FACS shows a remarkable alternative in production of high affinity and pure aptamers recognizing specifically cell membranes compartments (Jay 2013).…”