Selection Of Chloramphenicol DNA Aptamers And Establishment Of Aptasensor

Chloramphenicol (CAP), an effective and broad-spectrum antibiotic, inhibits both Gram-positive and Gram-negative bacteria and has been found to have potential lethal side effects on humans, such as leukemia. Therefore, many countries including USA, Canada, and China have banned the use of CAP in the food-producing animals. The European Union (EU) has also established a minimum required performance limit (MRPL) value for CAP at a level of 0.3×106 g·kg-1.Therefore, it is necessary to sensitively detect CAP residues in animal-sourced food.Aptamers, which are mainly SELEX (systematic evolution of ligands by exponential enrichment)-produced short oligonucleotides (ssDNA or ssRNA), can bind their target molecules with high affinity and specificity Because of their obvious features such as wider range of target molecules, better stability and easier modification aptamer-based detection strategy has been widely used in analytical chemistry, food safety and clinical diagnosis and treatmentmolecular recognition.In our work, CAP specific aptamers were used CAP-base as target and selected by in SELEX. Then an effective aptasensor based on real-time fluorescent quantitative PCR was built to detect CAP in milk.1?CAP-base was immobilized onto carboxylic magnetic beads. The Mag-SELEX system was developed for selecting several aptamers that can specifically bind with CAP by introducing ethanolamine as negative selection control. five potential candidate aptamers (i.e., No.1, No.4, No.5, No.6, and No.10) were selected through ten rounds of Mag-SELEX. These 5 candidate aptamers shared a low homology and high affinity. Their Kd values ranged from 0.03224±0.00819 to 0.3244±0.2293?M. No.4 and No.5 candidate aptamers with the lowest Kd value were selected for specific assessment and the results indicated that No.5 candidate had higher binding rate with CAP. According to Kd value and specificity result, No.5 could be the suitable aptamer of CAP and could be used as a recognition element in aptamer-based biosensors.2?we developed an effective aptasensor based on real-time fluorescent quantitative PCR. By optimization of the Experimental Conditions, probe 2 was chosen the optimum ssDNA probe, aptamer was added at a fixed concentration of 0.8?M and subsequent experiments were performed for parameter optimization, buffer A solution with 0.8M NaCl was chosen and 20 min was chosen as the incubation time in the experiment. The regression equation of the standard curve was Y=0.19487X+0.26348, with the correlation coefficient R2=0.98936. The linear range and the lower limit detection were 0.1?20 ng·mL-1 and 0.1 ng·mL-1.Moreover, this aptasensor system was further used to detect CAP in real spiked milk. The recovery rate of CAP from spiked milk samples ranged from 94.0% to 102.0%. These results indicated the developed aptasensor system in this study can be suitable for practical detection of residual CAP in milk.