Study On The Methods Of Real-Time Immuno-PCR For Detection Ractopamine

Ractopamine is a kind of β-receptor agonists. In recent years, it is used as animal feed additives frequently, for the purpose of improving the conversion rate of animal meat. However, the residue in animal will accumulate in human body through the food chain and bring the potential harm to human health. Some countries such as Countries of the Europen Union and Chinese have banned the addition of the substance in animal feed and drinking water.In the study, real-time immuno-PCR and immuno-LAMP were developed for detection RAC residue based on the method of enzyme linked immunosorbent assay(ELISA).The result are shown as follows:1. Ractopamine hapten(RAC-glutaric anhydride semialdehyde) was prepared using multiple anhydride method. Then it was coupled to carrier proteins to generate RAC-BSA, RAC-OVA by mixed acid anhydride method. Following the regular immunization protocol, New Zealand rabbits were immuned with RAC-BSA to obtain the anti-RAC polyclonal antibody with high titer.2. The RAC-HRP conjugate was synthesized by mixed anhydride method. Subsequently, the RAC-HRP and polyclonal antibody of RAC were used to established direct competitive ELISA method for determination of RAC. With the RAC concentration range of 0.15-5.0ng/m L, the IC50 value was 1.1ng/m L and the LOD(IC10) was 0.15ng/m L. The assay illustrated the high specificity with RAC, and the low cross reaction ratio with other 5 kinds of β-agonists compunds(CR<5.3%). In sample test, the recovery rates is acceptable ranged from 79%-94%. The coefficients of variation were in the range of 0.9-4.7% for inter-assay and 3.1-5.1% for intra-assay. Therefore, the method was proved to be well accuracy.3. The research utilized the real-time PCR as a means of signal detection to set up the real-time immuno-PCR assay based on ELISA. The antibody and report DNA labelled with biotin were prepared by using 5’-end with biotin of PCR primers and NHS method,respectively. The signal amplification of antigen and antibody binding was relied on the system of streptavidin and biotin. The key factors of the reaction,including the reaction carrier,labeled antibody and antigen concentration,avidin concentration, washing times and Bio-DNA were optimized. Finally, PCR tube with 0.6% Glutaraldehyde was chosen. After coating 2μg/m L detection antigen, the 0.326μg/m L Bio-anti-RAC p Ab were added. And then, 2.57μg/m L streptavidin and 0.35 ng Bio-DNA binded. In order to ensure the removal of unbind Bio-DNA, five times washing buffer and water should be conducted. After testing, the LOD of the method was 0.0039ng/m L, and IC50 was 0.18ng/?L. Compared with competitive ELISA, the method improved two order of magnitude The antibody showed high specificity with RAC and there was no cross reaction with other 16 kinds of β-agonist(CR<0.018%).The spiked recovery experiment result demonstrated the acceptable recovery rate of 80-120% within the concentration of 0.25-1.0ng/m L. The low inter- and intra- variation coefficient showed this method had well precision.4. The LAMP primers of Bio-DNA were designed. Optimizing the reaction condition to determine its reaction system: 0.2mmol/L F3&B3, 1.6mmol/L FIP&BIP,1.4mmol/L d NTPs,0.6M Betaine,8mmol/L Mg SO4,10mmol/L KCl,20mmol/L Tris-HCl, 10mmol/L(NH4)2SO4, 0.1%Tween 20, 8U Bst DNA Polymerase, 200?mol/L Calcein, 500?mol/L Mn Cl2, 2.5μL of template and add dd H2 O to 25μL. The results were detected with real-time PCR instrument using the channel of SYBR Green fluorescence. In the sensitivity test of the visual LAMP assay, the result was 104copies/μL. Applied the method to the immuno-PCR to detect nucleic acid, the LOD of ILAMP was 0.0079ng/ml and IC50 was 0.41ng/m L. Compared with competitive ELISA, the method improved one order of magnitude. The antibody showed high specificity with RAC and there was no cross reaction with other 16 kinds of β-agonist(CR<0.04%).In this study, the preparation of RAC-BSA and RAC-OVA was used in obtaining the polyclonal antibodies successfully Meanwhile, the direct competitive ELISA of RAC was established. Based on the research of ELISA, the real-time immuno-PCR was set up and evaluated the performance, such as the specificity and accuracy. At the same time, immuno-LAMP was explored probably.