In Vitro Screening Of SsDNA Aptamers Specific To Salmonella Serotype O8by SELEX And Their Preliminary Application

Objective：The objective of our research is to use SELEX(Systematic evolution ofligands by exponential enrichment) against the Salmonella serotype O8as a target tochoose a group of oligonucleotide aptamer with high affinity and specificity from aliborary contained35random sequences at total length78in vitro, for the rapiddetection of pathogenic microorganisms provide theoretical foundation andexperimental evidence.Methods:(1) It was using Salmonella serotype O8as a target and building a78nt ssDNAcontaining35nt random region in the middle flanked by defined sites library in vitro.After nine rounds of SELEX selection, the high affinity and specificity of theaptamers with Salmonella serotype O8were obtained and the counter-selectionagainst E. coli and S.choleraesuis were introduced in the4th and the7th rounds inorder to improve the specificity of the screening. According to Digoxigenin-anti-digo-xigenin-AP system we detected the binding rate between the product every one roundof selection and Salmonella serotype O8.(2) The nine rounds of selection product were amplified, purified, cloned andsequenced. Using Clustal W, RNA Structure, MEGA4and DNA MAN softwares wereanalyzed the aptamers of the primary structure homology and the predictionsecondary structure.(3) Extracted cloning bacteria plasmid and detected the binding combinationbetween aptamers and Salmonella serotype O8to selected a significantly higherabsorbance value of two cloned aptamers. Eventually we obtain an optimal clonedaptamer by the comparison of Kd value and specificity.(4) We used5’-FAM and5’-TAMRA labled the optimal cloned aptamer todirectly bind Salmonella serotype O8and counter-bacteria, wash and finalize, thenanalyzed by fluorescence microscope. Initially established a rapid identificationSalmonella serotype O8method by fluorescence labled aptamers.Results: (1) Results showed that the Absorbance values of the screening product withSalmonella serotype O8were increased from0.20to0.89, but the last two roundswere little difference, which indicating the binding ssDNA with Salmonella serotypeO8were not increasing when the binding sites were full.(2)22clones were randomly selected to be sequenced and the result is dividedinto3groups. Group A was of the same sequence as the expected fragment length,which consisted of19fragments and there are2pair aptamers sequence thathomology of up to100%. Group B consisted of2fragments was lack of one basesequence in the random region.Group C consisted of1fragments was increase3basesequence in the random region. Phylogenetic tree diagram shows the most homologywere more than70%up to100%. By RNA Structure software to predict the secondarystructure, the result is of the main stem-loop structure.(3) The absorbance values were significantly increased between cloned aptamerB10, G05with Salmonella serotype O8. The aturation determination showed that theKd value of B10was32.04nM and the Kd value of G05was175.9nM. Because Kdvalue is lower, the affinity of aptamer is more high, therefore we chose the optimalaptamer B10. When aptamer B10for the specific detection, we found that theabsorbance values of Salmonella serotype O8and B10aptamer was up to0.863,while E. coli and S.choleraesuis were0.251and0.375.(4) We used5’-FAM and5’-TAMRA labled the optimal cloned aptamer B10todirectly bind Salmonella serotype O8and counter-bacteria by fluorescencemicroscope, showing that the B10aptamer was binding to Salmonella O8, while E.coli and S.choleraesuis only a very small amount of the combination. Fluorescentlylabeled aptamer detected the ensitivity of Salmonella serotype O8was102/ml.Conclusions: The topic of our research is to applied SELEX against the Salmonellaserotype O8as a target to select oligonucleotide aptamer with high affinity andspecificity. After nine rounds of SELEX selection, the high affinity and specificity ofthe aptamers with Salmonella serotype O8were obtained and successful determinedthe best aptamer. Establish a rapid diagnosis method by fluorophore-labled aptamersfor the rapid detection of Salmonella serotype O8, providing a foundation forinvestigation and therapy infection of caused by Salmonella.