| Extensive use of tetracyclines in veterinary medicine for their application as antibiotics and growth promoters has lead to their accumulation in food products, such as meat, milk, and eggs/chicken and causes serious threat to human health. The most persistent and frequently found contaminated among the tetracyclines is oxytetracycline (OTC) because of its effective antimicrobial properties and low price.Specific detection of OTC is difficult by conventional techniques. Although enzyme-linked immunosorbent assay (ELISA) is the standard method to detect OTC residue in foods in most of countries, it is high costly due to its antibody preparation and production. Aptamer technology, a newly developed technique, is capable of binding target molecule with high specificity and affinity. Aptamer performances same characters as antibody with more additional advantages. The aim of this research was to establish an aptamer-based technology to detect OTC residues in food samples.Firstly the possibility of examining the binding efficiency of OTC with OTC-aptamer was investigated by electrophoretic mobility shift assay (EMSA). Results showed that EMSA cannot to be used for this purpose, although different binding conditions were tested in this study, such as the concentration of native polyacrylamide gel, ionic strength of binding buffer, and quenching conditions. Moreover, binding was not detected by EMSA when OTC-HRP and B-ATP-aptamer complementary strand was applied in the experiments, which confirmed that EMSA is unable to detect binding affinity for small molecules, such as OTC and ATP, with their aptamers.The Kd value of ATP-aptamer and OTC-aptamer were then determined as 1.12μM and 0.780μM respectively, by ultra-filtration and high performance liquid chromatography (HPLC). The change of aptamers'affinity was not significant (P>0.05) when they were modified by attaching biotin and Poly T. Ten per cent of whole milk could not influence ATP-aptamer Kd value significantly (P>0.05), but ATP-aptamer-PCS2 prevented the binding between ATP and ATP-aptamer significantly (P>0.05).Three conditions were optimized:coating streptavidin (SA) on microplates, fastening B-OTC-aptamer on SA pre-coated microplates and marking OTC with horse radish peroxidase (HRP). Different coating conditions such as coating buffers, SA concentrations, and various microtitration plates, were compared by testing the stabilities and coating-related variations (CV values). The optimized coating method was:microtitration plates (Jiete high-binding) were coated with 100μL of streptavidin (2μg/mL) in 0.05 mol/L carbonate buffer (pH 9.6) at 37℃for 16 h, and then the buffer was evaporated overnight and left SA on the surface of the wells. The capacity for B-OTC-aptamer of coated plates was 5.36 pmol per well, which was stable after incubated 7 days at 37℃, while the coefficient of variation was 2.95%. The aptamer-based ELISA technology was established. The sensitivity of it was low and the limit of detection of OTC residue was 1.512 g/L. Precision was investigated by variations of inter-assay and intra-assay with 10.32% and 16.46% respectively, and the recovery in whole milk was 71-84%. The sensitivity of this method did not match the commercial ELISA kit on the market, indicated that the aptamers affinity used in this study is improvable.
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