Selection And Primary Applied Research Of Aptamers Targeted To Enterohemorrhagic Escherichia Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7(E.coli O157:H7) is an important food-borne pathogens related to public health, it has posed serious public health hazard all around the world. Therefore, it is vital to realize the fast detection of E.coli O157:H7 in every field. Using the technology of Systematic Evolution of Ligands by an Exponential Enrichment(SELEX), a single-stranded DNA or RNA fragment named aptamer can be screened out from the random oligonucleotide library. Aptamer can form specific structures to bind targets with high specificity and affinity. Owing to its excellent characters with high sensitivity and high specificity, aptamer can be used as recognition elements in many fields of detection system.In this experiment, Subtractive Cell- SELEX technology was utilized to screen and obtained a single-strain DNA(ssDNA) aptamer which could bind to E.coli O157:H7 specifically. Based on above, magnetic nanoparticles were applied to establish a neotype detection system to test E.coli O157:H7 in water samples. First,an ssDNA pool composed by 88 bases was synthesized in vitro, the random part of ssDNA was composed by 45 bases including adenine(A), thymine(T), guanine(G),cytosine(C). Then using the Subtractive CELL- SELEX technology and taking E.coli O157:H7 as targets to carry out seven rounds of repeated screening. The products in sixth rounds which bound targets with highest efficiency were singled out for cloning sequencing. Softwares of Chromas and DNAMAN were applied to analyze family homology and simulation of the secondary structure of the sequencing results. The representative sequences were selected to synthesize and label FITC, and then used fluorescent chemical analyzer, flow cytometry and laser confocal microscope for affinity and specificity determination of aptamers. According to the above results, an ssDNA aptamer named A46 was obtained to recognize E.coli O157:H7 specifically.The Kd value of aptamer A46 is 82.45±18.46 nM. Biotin labeled Aptamer A46 was applied to combine streptavidin-conjugated magnetic nanoparticles via the strong streptavidin--biotin interaction. Taking this complex and FITC labeled A46 as captureand fluorescence probe, respectively. Finally, the method of fluorescence detection of E.coli O157:H7 in practical samples was established. Under the optimal experimental conditions, the detection linear range of E.coli O157: H7 was 90 ~ 5 x 104 cfu/ml, the lowest detection concentration was 88 cfu/ml, the detection rate of practical water added with E.coli O157:H7 was as high as 93.95%;among the practical food samples,the highest detection rate of E.coli O157: H7 was more than 80%.