In this paper, a Whole-Bacteria SELEX (WB-SELEX) strategy was adopted to isolate specific aptamers against Shigella sonnei. Real-time PCR amplification and post-SELEX experiment revealed that the selected aptmers possessed a high binding affinity and specificity for S. sonnei. Of the 21 aptamers tested, the C(t) values of the SS-3 and SS-4 aptamers (Ct = 13.89 and Ct = 12.23, respectively) had the lowest value compared to other aptamer candidates. The SS-3 and SS-4 aptamers also displayed a binding affinity (KD) of 39.32 ± 5.02 nM and 15.89 ± 1.77 nM, respectively. An aptamer-based fluorescent biosensor assay was designed to detect and discriminate S. sonnei cells using a sandwich complex pair of SS-3 and SS-4. The detection of S. sonnei by the aptamer based fluorescent biosensor platform consisted of three elements: (1) 5’amine-SS-4 modification in a 96-well type microtiter plate surface (N-oxysuccinimide, NOS) as capture probes; (2) the incubation with S. sonnei and test microbes in functionalized 96 assay wells in parallel; (3) the readout of fluorescent activity using a Cy5-labeled SS-3 aptamer as the detector. Our platform showed a significant ability to detect and discriminate S. sonnei from other enteric species such as E. coli, Salmonella typhimurium and other Shigella species (S. flexneri, S. boydii). In this study, we demonstrated the feasibility of an aptamer sensor platform to detect S. sonnei in a variety of foods and pave the way for its use in diagnosing shigellosis through multiple, portable designs.
A Gram-stain-negative, motile bacterium, designated strain YE3 T , was isolated from activated sludge obtained from a municipal wastewater treatment plant in Daejeon Metropolitan City, Republic of Korea. The cells were oxidase-and catalasepositive, and grew under aerobic conditions at 10-40 C (optimum, 30 C), with 1.0-8.0 % (w/v) NaCl (1.0 %) and at pH 5.5-9.0 (pH 7.0). Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain YE3 T was most closely related to Pusillimonas harenae KACC 14927 T (98.2 % sequence similarity) and Pusillimonas ginsengisoli KCTC 22046 T (98.0 %). DNA-DNA relatedness values for strain YE3 T and P. harenae KACC 14927 T , P. ginsengisoli KCTC 22046 T and P. soli KCTC 22455 T were 28.7±2.27 %, 21.3±1.16 %, and 14.0±0.67 %, respectively. The genomic G+C content of the type strain YE3 T was 59.3 mol%, as determined by whole-genome sequencing. The dominant fatty acids were C 16 : 0 (39.2 %) and C 17 : 0 cyclo (37.5 %). The major polar lipids of strain YE3 T were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Two aminophospholipids and four unidentified lipids were also detected. Furthermore, strain YE3 T was able to oxidize thiosulfate under heterotrophic conditions. Based on the phenotypic, genotypic, chemotaxonomic and phylogenetic analyses, strain YE3 T represents a novel species of the genus Pusillimonas, for which the name Pusillimonas thiosulfatoxidans sp. nov. is proposed. The type strain is YE3 T (=KCTC 62737 T =NBRC 113113 T).
Shigella sonnei isolate invasion plasmid antigen protein, IpaH, was successfully expressed in recombinant overexpression bacterial system. The soluble expression IpaH was enhanced with molecular chaperon co-expressed environment. Specific aptamer IpaH17 was isolated through the SELEX process and showed fM binding affinity. IpaH17-SPR biosensor platform was involved to verify the binding sensitivity and specificity. The IpaH concentration dependent IpaH17-SPR sensor response was highly linear with a linear regression constant of 99.4% in the range between 0 and 100 ng/mL. In addition, S. sonnei revealed the specific RU value and detected in a real-time manner within 1 hour. Our study indicated that IpaH17-SPR sensor can allow for rapid, sensitive and specific determination of Shigella sonnei virulent factor.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.