| Brucellosis is a zoonotic disease caused by Brucella. It is one of the zoonotic diseases which could seriously endanger the safety of public sanitation around the world. Brucellosis poses immensely potential threaten to the production of animal husbandry and the human health. The research primarily focuses on the parts of Brucella's outer membrane proteins (omp), aiming at developing the multiplex PCR pathogen method and ELISA antibody method for the detection of brucellosis and carrying out their preliminary application. Our purpose is to hopefully utilize the methods in the rapid detection and diagnosis of brucellosis in clinic.1. Three specific pairs of primers for the same pathogen were designed according to the omp31(FJ984569), bp26(AY166766) and omp10(BRUOMP10A) gene sequences published on the National Center for Biotechnology Information (NCBI) Genbank database. Through the optimization of the experiment conditions, a rapid multiplex PCR detection method was developed. The results showed that the lowest amount of DNA which could be detected was 1.1×10-3ng. The minimum number of bacterium detected from the modeling samples of milk infected with Brucella was 80 cfu/ml. 484 milk samples collected from several dairy farms in Shaanxi province were detected by the method. The result showed that there were 6 positive samples and the positive detective rate was 1.24 %. The detection result indicates that this method has the advantages of specificity, sensitivity and repeatability, so it can be used in the clinical detection of dairy cattle brucellosis and its epidemiological surveillance.2. A polymerase chain reaction (PCR) method was used to amplify the omp10, omp25 and bp26 gene regions, which were ligated with pET-32a prokaryotic expression vector into recombinant plasmids. The recombinant plasmids were transformed into Escherichia coli BL21(DE3) PlysS whose expression was induced by IPTG and then separated and purified through HisTrap HP affinity column. The results of gene sequencing and restriction enzyme digestion identification both proved that the pET-32a-omp10/omp25 vectors were successfully constructed. The SDS-PAGE electrophoresis showed that the omp10 and omp25 recombinant proteins were both highly expressed in the form of inclusion bodies in E. coli and the bp26 recombinant protein was expressed in the soluble way. After the purification of HisTrap HP affinity column, recombinant proteins in size of 34ku, 44ku and 48ku were successfully obtained, which correspond to the expected protein molecular weight. The detection of western-blot proved that purified omp10, omp25 and bp26 recombinant proteins could be identified by the Brucella positive serum from immunized cattle.3. ELISA plates were coated respectively by the purified Brucella omp10, omp25 and bp26 recombinant proteins that act as antigens. The ELISA antibody method for the detection of Brucella was developed through the optimization of the reaction conditions. The results showed that the concentration of the coated omp10, omp25 and bp26 antigens was 10μg/mL, 5μg/mL and 5μg/mL and the best condition was under 4℃overnight. The dilution multiple of serum was all 1:100. The optimal dilution multiple of HRP secondary antibody was all 1:5 000. The positive and negative judgment of S/P critical value of omp10, omp25 and bp26 was 0.261, 0.192 and 0.191 respectively. Based on the above results, the indirect ELISA method, tube agglutination test and rose bengal plate agglutination test were compared to detect 432 bovine serum samples. The results were that those samples detected positive by SAT and RBPT also showed positive by ELISA, while those samples detected positive by ELISA showed partially positive by SAT and RBPT. Therefore, the ELISA method was more sensitive than SAT and RBPT. The experiment results prove that the established ELISA detection method own the characteristics of specificity, sensitivity and rapidity, which are suitable for the rapid diagnosis of brucellosis in clinic. |
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