Abstract
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.
Highlights
The Shigella species of bacteria is the causative agent of shigellosis, known as bacillary dysentery, which has symptoms ranging from dysentery, tenesmus, and abdominal cramps to diarrhea with blood and mucous [1]
Whole Bacteria Cell Systematic Evolution of Ligands by exponential enrichment (SELEX) for the Isolation of S. sonnei Specific DNA Aptamers
After 10 rounds of SELEX, nanodrop spectrophotometer analysis was performed to monitor and assess the binding affinity of aptamer sub-pools to S. sonnei
Summary
The Shigella species of bacteria is the causative agent of shigellosis, known as bacillary dysentery, which has symptoms ranging from dysentery, tenesmus, and abdominal cramps to diarrhea with blood and mucous [1]. It is estimated that more than 165 million cases are reported annually and approximately one million people die from shigellosis worldwide each year [2]. The Shigella genus is comprised of four species: Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei. S. flexneri is the most common species in developing countries whereas S. sonnei is the predominant species in industrialized countries [3,4]. Several reports have shown that most shigellosis outbreaks are closely related to an influx of people with a history of international travel, which is difficult to Molecules 2017, 22, 825; doi:10.3390/molecules22050825 www.mdpi.com/journal/molecules. Since shigellosis tends to spread very rapidly by mass feeding in closed and concentrated groups, such as army barracks and schools [7], there is an urgent need for rapid, simple, and specific detection methods for shigellosis
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