Application Of Polymerase Chain Reaction Assay For Detecting Of Brucella And Study Of Molecular Typing Of Brucella

In order to mark up for a dificiency in the appoach of microbiology and serological detection of Brucella. To explore and establish a rapid method of molecular typing of Brucella. The study is started by following three aspects of research.First of all, To analyze a great number of partition Brucella of types sequence which have been issued as specific genotype, Searching for the distributing role of genotype- specific nucleotides, designing the specific primers, applying PCR method to identify specific type of Brucella. Established PCR-SSCP method distinguishes vaccine strain A19 from other specific types of Brucella.Secondly, The downstream sequence of VirB7 to upstream sequence of VirB9 gene from type IV secretion system(TTSS)of brucella was used as template and the primers were designed. The extraction methods of DNA were optimized. Established IR-PCR (Internally Reference PCR) method of assay were used for detection of Brucella in anticoagulated blood and milk.Finally, The PCR kit for detecting VirB8 gene of Brucella in blood was assembled and tested for its specificity, sensitivity, storage life and stability. Use the PCR kit, the blood samples from Xinjiang and Shanxi provinces of which were detected.The results show that the VirB8-PCR,AMOS-PCR,B1-PCR method can be used to diagnosis of Brucellosis and identify specific type of Brucella; PCR-SSCP assay can be detected vaccine strain A19. 2. Established IR-PCR (Internally Reference PCR) method of assay is more effective to avoid operational errors of process of DNA extraction, of which the positive coincident rate of the IR-PCR detecting result with purification identification was 100 % and the positive coincident rate of the IR-PCR detecting result with iELISA was 62.5%; The detection limit was as low as 35CFU/ml for blood and 350CFU/ml for milk. The IR-PCR are very suitable for detecting brucella infection of anticoagulated blood and milk in laboratory. 3.The 2 samples showing negative by SAT were proved Brucella positive by the kit , the sensitivity of the kit being higher than that of the SAT. The detection limit was as low as 35CFU/ml for PCR kit, which revealed that the specificity of the kit is very reliable in -20℃for 6 months.