| Salmonellosis is one of the most significant zoonoses on the field of public health, and its pathogenic salmonella are enterobacteriaceae. It is the general terms of a large group of gram-negative bacillus which are parasitic in the gut of humans and animals and are similar in the antigen structure and biochemical reaction. Especially, pathogenic bacteria represented by Salmonella typhimurium have posed a great threat in the food safety, environmental monitoring, and disease prevention, and caused great harm to human health. How to realize the goal of rapid detection of salmonella has been becoming the current hot issues.Aptamer is a short single-stranded DNA or RNA sequences which is abtained by systematic evolution of ligands by exponential enrichment (SELEX). It has both of high affinity and strong specificity in combination with the corresponding target molecules such as metal ions, organic small molecule drugs, proteins, and even the entire cell. Having above good characters, they can be widely used in the detection of diagnosis and biological molecular analysis.Graphene is a new type of two-dimensional nanomaterial with a single honeycomb mesh structure packed tightly with pure carbon atoms. The larger specific surface area, excellent thermal conductivity and electrical conductivity, make graphene widely used in many fields. Being one of the important graphene derivatives, Graphene Oxide (GO) has good affinity and solubility in water as its oxygen groups existing in the surface. Recently, by using the good biocompatibility, high fluorescence quenching efficiency and large surface area of GO, many fluorescent biosensors have been developed.In this thesis, by selecting S. typhimurium as the target, we carried out the research work focused on highly sensitivity and selectivity pathogen detection. The main works are summerized as follows:1. The establishment of SELEX screening method of aptamer for S. typhimurium.Taking S. typhimurium as the target, we built a total length of78bases library containing35single nucleotide bases random sequence by SELEX in vitro. According to11rounds of repeatedly regular selections and4rounds of subtractive SELEX with S. enteritidis, S. cholerae-suis, S. Paratyphi A and S. Arizonae as targets, a nucleic acid aptamer group specifically binding with S. typhimurium are primarily selected. We got the39aptamer sequences by the cloning sequencing of the PCR products for the final round of selection. Then we take the homology analysis of their primary structure and the simulation analysis of their secondary structure, and finally obtained6sequences having high affinity and specificity with S. typhimurium. The binding affinities of the aptamers were determined through fluorescence assay. Their Kd values were calculated and up to0to100nM range, and the aptamer Apt6has the lowest Kd (30±4nM). The binding specificities of the selected aptamers to target bacteria were also tested, and the results clearly demonstrated that the aptamer Apt6could detect S. typhimurium with high specificity and had no cross-reaction with other Salmonella serovars and pathogens. 2. Fluorescent aptasensor for the determination of Salmonella typhimurium based on a GO platform.With the fluorescence quenching property of GO and the property that aptamer can specifically recognize its target, we built a rapid, simple, high sensitive fluorescence aptasensor. In the absence of target, the fluorescence was fairly weak as result of the FAM-labeled aptamer adjacent to GO. If, however, the fluorophore is released from the GO due to the formation of the target/aptamer complexes, fluorescence intensity is substantially increased. Under the optimum conditions, the assay displays a linear response to bacteria in the concentration range from103to108cfu/mL, with a detection limit of l00cfu/mL. The method is selective in that fluorescence is not much enhanced in case of other bacteria. Thus, this aptasensor based on GO is believed to possess a large potential with respect to the rapid detection of bacteria. |
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