The Methods Of Ractopamine Determination And DNA Purification Using Magntic Paticles As Carriers

Magnetic particles are composed of magnetic nanoparticles and inorganic/organic materials via their interactions such as coating, crosslinking and modification. These composite particles are super-paramagnetic, with large surface and functional groups. The antigen/antibody, enzyme, DNA/oligonucleotides and drugs can be immobilized on their surface, so magnetic particles have been widely used in immunoassay, nucleic acids purification, and cell sorting and drug delivery. Using two kinds of magnetic particles (GoldMag particles and carboxyl-terminal silanized magnetic particles) as carriers, the methods of ractopamine determination and the DNA purification from biological samples was set up respectively in this thesis.GoldMag are particle complexes composed of magnetic particles as the core component with gold nanoparticles chemically assembled on the magnetic core surface, they offer advantages in both magnetic separation and immobilization of biomolecules. The conjugate of ractopamine and ovalbumin (RCT-OVA) was coupled on the surface of GoldMag to quantitatively analyze the ractopamine residue in animal urine, feed and tissues based on the indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). A calibration curve of ractopamine was established and the limit of detection is determined, the recovery and the cross reactivity of the monoclonal antibodies for other 6-agonists was tested using this method. The result showed that the linear range of the RCT concentrations was 1～100 ppb and the relative coefficient was 0.993, the detection limit was 0.668 ppb, the whole time of the experiment was less than four hours and the rate of recovery of ractopamine was range from 72.6 to 106.2%, the coefficient of variation was no more than 13.4%. The method improved the sensitivity of detection and reduce the time of process compared with the conventional sandwich ELISA using microplate as carrier and can be used to detect the ractopamine in urine, feed and tissues. Genomic DNA from the whole blood and saliva was purified using the carboxyl-terminated magnetic particles separately. The experiment conditions such as the kinds of the magnetic particles, amount of the magnetic particles, lysis buffer and the ratio of amount of magnetic particles and binding solution were optimized. A small-scale purification system from 200μL of whole blood was set up. The method for isolating DNA from saliva was also studied; the results indicate that about 3～7μg of genomic DNA was obtained from 200μL whole blood using 0.1 mg of magnetic particles. The ratio of OD₂₆₀/OD₂₈₀ was between 1.7 and 1.9 and the CV value was lower than 8%. The genomic DNA yield was from 0.5 to 4μg from 200μL saliva using 0.1 mg magnetic particles. A method for purification of the plasmid DNA from bacteria using carboxyl-terminated magnetic particles was also set up. E.coli cell was treated with alkaline lysis and the purification process for 1 mL overnight bacterial suspension was optimized. 0.2 mg of magnetic particles was selected to isolate the plasmid DNA and the yield was between 3 and 12μg, the ratio of OD₂₆₀/OD₂₈₀ was between 1.7 and 1.9, the CV value was lower than 8%. Compared with the traditional method, this method has the advantages such as rapid performance, easy operation, no organic solvents etc. DNA purification method provides potential commercial value in the near future.