| Abstract (English): |
Florfenicol (FFC) is embryotoxicity toxic, and its residues in food animal tissues can seriously harm the health and safety of consumers. In order to establish a method for rapid detection of FFC residues in animal food, the upstream and downstream primers were designed according to the primer design principle. A random single stranded oligonucleotide (ssDNA) library with the length of 80bp was constructed according to the primer sequence. The 3' and 5' ends were fixed 20bp primer binding sites, with a random sequence of 40bp in the middle. Through GO-SELEX screening, the pretreated ssDNA library was coincubated with equal moles of FFC and GO. Through GO π- π the conjugated bonds adsorbed ssDNA that did not bind to the target FFC, while the ssDNA that had bound to the target (FFC ssDNA) was left in the supernatant, then the FFC ssDNA complex was obtained by centrifugation. After centrifugation, extraction and alcohol precipitation, the ssDNA obtained from FFC ssDNA complex in each round of the screening was used as template for PCR amplification. The double stranded DNA (dsDNA) of PCR product was completely split into single strand by streptavidin, which was used as secondary ssDNA library to enter the next round of screening. The recovery rate was calculated once after each round of screening. When the change of recovery rate tended to be stable, the antiscreening products of chloramphenicol (CAP) and thiamphenicol (TAP) were introduced to remove the ssDNA with the poor specificity for FFC. When the recovery reaches saturation, screening stops. Finally, 12 rounds of aptamer screening were carried out according to the calculation results of recovery rate, of which 10 rounds were positive screening, and the seventh and eleventh rounds were reverse screening. PCR results of ssDNA obtained from 12 rounds of screening showed that the desired ssDNA fragments were obtained through 12 rounds screening. Forty-six different aptamer sequences were obtained by cloning and sequencing the products from the ninth and twelfth rounds. Based on the secondary structure, free energy (△G) prediction and phylogenetic analysis of 46 nucleotide sequences, eight candidate aptamers were obtained, including A1, A26, A27, A31, B18, B28, B30 and B37. The dissociation constants (Kd values) of eight candidate aptamers were determined to analyze their affinity with FFC. The results showed that the Kd values of the eight candidate aptamers ranged from 11.811 nmol/L to 54.097 nmol/L; The results showed that both aptamers could specifically bind to FFC, and the specific binding effect of B18 to FFC was stronger, so it could be used as the specific aptamer of FFC. In this study, the aptamers of florfenicol were screened by SELEX for the first time, which laid a foundation for the rapid detection of florfenicol residues in animal food. [ABSTRACT FROM AUTHOR] |