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.

Highly Sensitive Colorimetric Biosensor for Staphylococcal Enterotoxin B by a Label-Free Aptamer and Gold Nanoparticles

“…Only after the addition of SEB2 aptamer or random DNA a similar characteristic SPR absorption band of AuNPs at 520 nm appeared. We consider the surface of AuNPs was absorbed by ssDNA to protect the AuNPs from salt-induced aggregation (Lavu et al, 2016 ). However, after addition of SEB, a SPR band at about 620 nm was clearly observed and the color of the AuNPs solution changed from red to purple (Figure 2C ).…”

“…Hence, the development of aptamer-AuNPs colorimetric biosensors can be a simplified, attractive alternative detection system. Many researchers extended the target to various analytes, such as protein (Zhang et al, 2010 ), metal ion (Li et al, 2009 ; Wang et al, 2010 ) small molecule (Xu et al, 2009 ) and bacterial cells (Lavu et al, 2016 ) employing aptamers as recognition and AuNPs as a colorimetric indicator. However, we were unaware of any exclusive study discussing on aptamer-AuNPs based colorimetric assay for detection of SEB.…”

Nucleic acid aptamer-based methods for diagnosis of infections

“…3C). In addition, aptamer-based colorimetric strategies for the detection of pathogenic cells have also been developed (Wu et al, 2012;Bayraç et al, 2017;Bruno, 2014;Yuan et al, 2014aYuan et al, , 2014bHu et al, 2017;Lavu et al, 2016). Consequently, the presence of free protamine is detected by a potential change of a polycation-sensitive membrane electrode.…”

Oligonucleotide aptamers: promising and powerful diagnostic and therapeutic tools for infectious diseases

“…28 Several groups have used whole-cell SELEX to identify ss-DNA aptamers binding to S. Typhimurium, Salmonella Paratyphi and Salmonella enterica serovar Enteritidis ( S. Enteritidis) for Salmonella detection. [29][30][31][32][33][34][35] These aptamers binding to the whole cell of Salmonella were conjugated to nanomaterials such as gold nanoparticles (AuNPs) and fluorescent nanoparticles as probes, magnetic nanoparticles (MNPs) as concentration elements, or loaded onto SWCNTs and reduced graphene oxide (rGO). 29 , 30 , 34 , 36 , 37 Oh et al immobilized AuNPs-ssDNA aptamer conjugates onto a localized surface plasmon resonance (LSPR)based sensor chip and reported a method for detection of S. Typhimurium in spiked pork meat samples with the detection limit of 1.0 × 10 4 CFU/mL.…”