The Selection And Application Of Aptamers Against Lean Meat Powder Based On Library Immobilization

Clenbuterol hydrochloride(CLB), salbutamol(SAL) and ractopamine(RAC) are most widely-used additives in livestock breeding, and are the primary animal forbidden drugs in the directory of our country. In recent years, the acute poisoning events caused by CLB, SAL and RAC took place frequently, which brought great harms to social stability and human health. Thus, it is urgent to develop rapid and sensitivity analysis methods applied in on-site detection for CLB, SAL and RAC. In this study, aptamers binding against CLB, SAL and RAC respectively were obtained based on ssDNA library immobilized SELEX technique. The selection based on library immobilized has advantages of low cost and simple operation. It also eliminate the side effect of steric hindrance due to target immobilization, and achieve simultaneous selection against multiple targets. Aptamers selected by this method can used as novel recognition probes applied in on-site detection. The process were as follows:Firstly, the selection of aptamers against CLB, SAL and RAC based on library immobilization SELEX. The biotin labeled complementary strand P1(Biotin-P1)were designed to paring with the primer of ssDNA library. After complementation, ssDNA library were immobilized on the magnetic beads through Biotin-P1. The immobilized ssDNA library were incubated with CLB, SAL and RAC. When ssDNA bind to targets, ssDNA will change the structures leading to separating from Biotin-P1 and releasing from magnetic beads to the solution. ssDNA binding to targets were obtained by magnetic separation. By repeating ssDNA library immobilization, incubation, separation, elution, PCR, digestion and purification, the aptamers binding against CLB, SAL and RAC respectively were enriched after sixteen positive selection rounds and seven negative selection rounds.Secondly, the identification of affinity and specificity. after cloning and sequencing, the forty sequences against each target were obtained. Based on the analysis of homology and secondary structures, eight aptamer candidates for CLB, ten candidates for SAL, and nine candidates for RAC were synthesized with FAM label and used for identification. The results indicated that aptamer Apt-3 can bind to CLB and SAL simultaneously, and the dissociation constant(Kd) values were 38.45±7.01 nmol/L and 47.00±6.42 nmol/L respectively. Aptamer CLB-2 can bind to CLB specifically, and Kd values was 76.61±12.70 nmol/L. Aptamer SAL-5 can bind to SAL specifically, and Kd values was 53.60±11.81 nmol/L. Aptamer RAC-6 can bind to RAC specifically, and Kd values was 54.22±8.02 nmol/L.Finally, the fluorescent analysis method was developed by employing aptamers as molecule recognition elements for CLB, SAL and RAC. When aptamer CLB-2 recognized CLB specifically, a linear relationship(R2=0.9905) was obtained ranging from 0.10 to 50 ng/m L with LOD of 0.08 ng/mL. When aptamer SAL-5 recognized SAL specifically, a linear relationship(R2=0.9966) was obtained ranging from 0.50 to 100 ng/m L with LOD of 0.12 ng/m L. When aptamer RAC-6 recognized RAC specifically, a linear relationship(R2=0.9925) was obtained ranging from 0.10 to 100 ng/m L with LOD of 0.04 ng/m L. This method can be used in pork samples detection with the recovery rate ranging from 84.50% to 110.50%.