Screening And Application Of Three Veterinary Drug Residue Aptamers In Animal Food

Nowadays,the problem of veterinary drug residue has attracted the general attention of the general population,which can not only directly threaten animal and human health,but also affect the development of Chinese breeding industry and the progress of the international market.Chlorpromazine,amoxicillin and salafloxacin are commonly used veterinary drugs,indicating the detection of these three veterinary drug residues in animal food are very necessary.Traditional chromatography has the advantage of accurate results,but requires large instruments and pretreatment is complicated;immunoassay has high sensitivity and specificity,but the antibodies are not easy to prepare.The aptamer is a stretch of oligonucleotide that binds specifically to the target.Because of its in vitro synthesis,easy to modify,wide application range and other advantages,aptamers are widely used in the field of food safety analysis.Capture-SELEX selected the candidate aptamers for chlorpromazine,amoxicillin and salafloxacin.Further analysis of the binding properties of the candidate aptamers was performed to obtain aptamers with high affinity and high specificity.The binding mechanism of aptamer and target was also explored.Based on this,methods to detect chlorpromazine,amoxicillin and salafloxacin were constructed based on aptamer recognition combined with fluorescent and colorimetric methods.Details are as follows:First,high-affinity,high-specific aptamers were screened for chlorpromazine,amoxicillin and sarafloxacin using Capture-SELEX with immobilized libraries.The characterization of the screened secondary libraries and PCR products was verified by UV-visible spectrophotometer and gel imager,and the library enrichment was monitored using the fluorescence method during the screening process.Through ten rounds of screening,three kinds of veterinary drugs were obtained.Through high-throughput sequencing technology,based on the base order and frequency of the obtained sequence,combined with the sequence free binding energy and secondary structure information,seven candidate aptamers of chlorpromazine,amoxicillin,and sarafloxacin were selected,respectively.Secondly,the binding properties of chlorpromazine,amoxicillin and salafloxacin aptamers were analyzed based on isothermal titration（ITC）and Sybr Green I（SGI）fluorescence competition methods,respectively.By fitting the binding saturation curve,the aptamers CHL-3,AMO-1,and SAR-2 with high specificity for chlorpromazine,amoxicillin,and salafloxacin,which were 69.80±9.81 nmol/L,71.88±15.31 nmol/L,and 76.66±16.76nmol/L,respectively.Further examination of the specificity of the three aptamers showed that the aptamer has low binding forces with the corresponding target analogues.Third,the aptamer,target and binding mechanisms were explored by circular dichroism and molecular docking simulations.Circular dichroism results showed that all three aptamers were type B DNA,with enhanced base stacking effect after binding between the aptamer and the target,and the original conformational changes.The molecular docking simulation results showed that chlorpromazine mainly interacts with the base sites of A13,G14,G15,T16,C70of CHL-3,and G9,C10,T11,A12,A13,A43,C44 of AMO-1,and C37,T39,T58 and G59 of SAR-2.Finally,based on the high affinity and high specificity aptamers,the methods for chlorpromazine,amoxicillin and salafloxacin were established.Including the detection of chlorpromazine with Uio-66-NH₂ sealed with rhotamine 6G gated by aptamer CHL-3.The linear range ranges from 1 nmol/L to 100 nmol/L,The detection limit was 0.67 nmol/L.A colorimetric assay using the aptamer AMO-1 construct of horseradish peroxidase-catalyzed TMB coloration to detect amoxicillin.The linear range ranged from 5 nmol/L to 300 nmol/L,the detection limit was 2.41 nmol/L.Salafloxacin was detected using SGI fluorescence competition using aptamer SAR-2 constructs.The linear range ranged from 1 nmol/L to 50nmol/L,the limit of detection was 0.83 nmol/L.The above methods have been successfully applied to the detection of veterinary drug residues in eggs and milk samples,providing a new method for the detection of chlorpromazine,amoxicillin,and salafloxacin in food.