Selection And Sequence Optimization Of Tobamycin Specific Single Stranded DNA Aptamers And Their Applications

Tobramycin is an aminoglycoside antibiotic,which exhibit excellent antibacterial effect to a variety of Gram-negative bacteria.However,the abuse of tobramycin will lead to the residue in animal-derived foods,which will be harmful to human health through the food chain.Therefore,the establishment of efficient and rapid detection of tobramycin in food is of great importance.In this thesis,through magnetic beads-based exponential enrichment of ligand system evolution(SELEX),ss DNA aptamers specific to tobramycin were selected from the 79-nucleotides initial library containing 35 random sequence in the middle.After ten rounds of screening,the affinity between tobramycin and the sequences in the selected pool reached the highest.The screening product of the last round was amplified by PCR.After the transformation,37 positive clones were picked and sequenced.The nucleotide sequences were analyzed by DNAMAN and divided into nine families.The secondary structure of these aptamers were analyzed by Mfold online software.Among them,the secondary structures of four aptamer sequences(No.1,No.11,No.12,No.32)were highly similar.The dissociation constant(Kd)values of these four aptamers were determined by fluorescent method,which were 149.00 nmol·L-1,96.27 nmol·L-1,101.40 nmol·L-1 and 52.32 nmol·L-1,respectively.No.32 aptamer possesses the highest affinity for tobramycin.Then its specificity was verified,and the aptamer showed strong binding toward tobramycin,whereas very weak binding towards other antibiotics.The No.32 aptamer was modeled by Autodock 4.0 software with tobramycin.The results showed that nucleotides of 14-18 and 26-29 in the sequence plays important role in the interaction between the aptamer and tobramycin.Then the structure of No.32 aptamer was optimized by truncation.The number of bases of the two obtained truncated aptamers(No.32-1 and 32-2)were 49 nt and 34 nt,with Kd values of 48.27 nmol·L-1 and 58.92 nmol·L-1,respectively.The truncated 32-2 aptamer had a shorter sequence,and similar Kd value to the original sequence.Therefore,the 32-2 aptamer was chosen as the best aptamer for tobramycin.With the best aptamer 32-2,designed a method for the determination of tobramycin combined with gold nanoparticles(AuNPs)-based colorimetric method.The optimum concentration of NaCl was 120 mmol·L-1 and the optimum concentration of aptamer was 150 nmol·L-1.In the range from 200 nmol·L-1 to 1200 nmol·L-1,the absorbance of AuNPs solution at 520 nm was linearly decreased with the increased concentration of tobramycin.The specificity of this method was verified,which showed high specificity for tobramycin.The method was applied to detect tobramycin in honey samples.The color change of the AuNPs solution,the change of the absorbance at 520 nm and the linear relationship are consistent with the trend in the standard tobramycin,indicating that the matrix in 10 times diluted honey sample has little effect on the test results.Thus the established detection method can be applied to detect tobramycin residue in the real food samples.