title: Selection and Characterization of Aptamers Using a Modified Whole Cell Bacterium SELEX for the Detection of Salmonella enterica Serovar Typhimurium.
abstract: This study describes the selection of single-stranded DNA (ssDNA) aptamers against Salmonella enterica serovar Typhimurium using a modified whole cell systematic evolution of ligands by exponential enrichment (whole cell SELEX). For evolving specific aptamers, ten rounds of selection to live Salmonella cells, alternating with negative selection against a cocktail of related pathogens, were performed. The resulting highly enriched oligonucleotide pools were sequenced and clustered into eight groups based on primary sequence homology and predicted secondary structure similarity. Fifteen sequences from different groups were selected for further characterization. The binding affinity and specificity of aptamers were determined by fluorescence binding assays. Aptamers (SAL 28, SAL 11, and SAL 26) with dissociation constants of 195 ± 46, 184 ± 43, and 123 ± 23 nM were used to develop a nanogold-based colorimetric detection method and a sedimentation assay. The former showed a better sensitivity limit of 10(2) CFU/mL using aptamer SAL 26. This approach should enable further refinement of diagnostic methods for the detection of Salmonella enterica serovar Typhimurium and of other microbial pathogens.
authors.affiliation: Defence Food Research Laboratory (DFRL) , Mysore 570011, India.
authors.forename: Padma Sudha Rani
authors.initials: PS
authors.lastname: Lavu
authors.affiliation: Defence Food Research Laboratory (DFRL) , Mysore 570011, India.
authors.forename: Bhairab
authors.initials: B
authors.lastname: Mondal
authors.affiliation: Defence Food Research Laboratory (DFRL) , Mysore 570011, India.
authors.forename: Shylaja
authors.initials: S
authors.lastname: Ramlal
authors.affiliation: Defence Food Research Laboratory (DFRL) , Mysore 570011, India.
authors.forename: Harishchandra Sreepathy
authors.initials: HS
authors.lastname: Murali
authors.affiliation: Defence Food Research Laboratory (DFRL) , Mysore 570011, India.
authors.forename: Harsh Vardhan
authors.initials: HV
authors.lastname: Batra
mesh_terms: D052157:Aptamers, Nucleotide / Q000302:isolation & purification*; D001431:Bacteriological Techniques; D001483:Base Sequence; D004277:DNA, Single-Stranded; D052156:SELEX Aptamer Technique / Q000379:methods*; D012486:Salmonella typhimurium / Q000302:isolation & purification*
chemical_list: D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded
keywords: DNA aptamer; Salmonella Typhimurium; fluorescence; gold nanoparticle; whole cell SELEX