| Classical swine fever (CSF) is a highly hazardous infectious disease which caused by Classical swine fever virus (CSFV). The clinical features of CSF are highly-contact and rapid dissemination. During the modern farming, due to the vaccines inappropriate use and mutation in viruses. Disease in farms shift from large-scale popular local distributed, in pigs, the disease appears more like atypical chronic and recessive,The recessive carrier became the major trend of the CSF and usually found in the sow herd, which results the reproductive failure (such as abortions, stillbirths, mummified and so on) and have a damaging effect on pig industry. In this study, a direct immunofluorescence assay is established to provide a fast and sensitive reagent for the detection of classical swine fever virus. Specific research contents are as follows:1. Preparation of fluorescent antibody to classical swine fever virusRecovery and expansion culture of AH9strains which developed by the laboratory. It was the major protective antigen E2envelope protein for the swine fever, and then we used the Babl/c mice by intraperitoneal injection of hybridoma to prepare large number monoclonal antibody. The acid-ammonium sulfate and Sephadex G-25were used for antibody purification. We obtain a highly-purified monoclonal antibody with concentration up to11.491mg/ml. Two clear bands in the25kDa and50kDa can be seen by SDS-PAGE electrophoresis, it proof that the swine fever monoclonal antibody has a high purity. Then we get the monoclonal antibody titer up to1:51200by the ELISA. Then we use dialysis method to combine the antibody with fluorescein isothiocyanate (FITC). Finally, we successfully established a rapid direct immunofluorescence assay to detect CSFV.2. The best test conditions of the Classical swine fever virus monoclonal fluorescence antibodyWe optimized the experiment conditions, which including the fixing agent and time, the antibody concentration and incubation time, the antibody specificity and sensitivity, the antibody comparative and repeatability and storage life test and so on. According to the experimental results, the optimum conditions are set as follows:the sample is fixed with60%acetone and40%ethanol under-20°C for30minutes; antibody optimal working concentration was diluted by1:300; preferred antibody incubation time is60minutes. This method is specific, having no cross-reactivity with other common original. Sensitivity experiments show that the Real-time PCR method has two sensitivity levels than the FA, and the RT-Nested method has one sensitivity level than the FA. These results indicated that FA method has a high sensitivity. Direct immunofluorescence assay and laboratory known samples were used to detect the RT-Nested coincidence rate comparison. We found the coincidence rate of two methods was88.42%, which was a highly coincidence rate. We also confirmed of the reproducible of this method. Finally, we stored the fluorescent antibody in-20℃for6months and then tested the antibody sensitivity and compliance. The results showed that the fluorescent antibody still has a high specificity and sensitivity. All the results indicated that the new method is fast, sensitive and specific.2. The clinical application of swine fever fluorescent antibodyWe used the CSF fluorescent antibody to detect the frozen pathologic tissue section. The results show that the CSF fluorescent antibody method is highly matched with the RT-PCR method. Then we used the CSF fluorescent antibody method to detect463pig pathologic tissue samples from pig farms Sep-Nov,2012. We found62positive diseased samples in them and the positive rate was13.39%. |
PDF Full Text Request