Established Fluorescent Quantitative Detection Of Brucella And Immunochromatographic Strip For Diagnosis Of Brucella Serum

Brucellosis is a zoonotic disease caused by the genus Brucella, gram-negative, intracellular bacteria. It is endemic in various parts of the world, especially in China of the Xinjiang, Qinghai, Tibet and Mongolia province. The disease results in considerable economic losses and represents an important human and animal health problem Accurate and timely identification of infected individuals is critical for treatment and control. A lot of molecular diagnostic methods such as PCR and RT-PCR have been designed according to specificity of each Brucella spp.and the sensitivity, specificity were desired. Moreover, with the use of some new immunizing technology in medical science, some new diagnostic methods based on detecting antigen or antibody of Brucellosis have been futher studied.According to the nucleotide sequences of Brucella 16M published in GenBank. The bp26 gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pGM-T vector and then sequenced. The prokaryotic expression vector pET28a-BP26 was constructed by using the purified BP26 gene that was subcloned into the expression vector pET28a. The resultant pET28a-BP26was transformed into E.coli BL21 cells and induced by IPTG. The result of SDS-PAGE and Western-blot indicated that an approximately exogenous protein was observed. The expression protein was purified with Ni-NTA Agaros, and the purified pET28a-BP26 fusion protein was obtained. These results provided a basis for futher study of Brucellosis diagnosis.Established a method of colloidal gold immunochromatographic to diagnose the antibody of Brucella. immunochromatographic strip was prepared with colloidal gold sign SPA and Brucella BP26 protein. We detected the specificity, sensitivity and reproducibility of this method. Results The strip show two lines when detected positive Brucella blood serum, Only the control line colouration when detected others positive blood serum, the result could be observed by eyes in five or ten minutes The strip could detected positive blood serum of sheep that deliquated 1:100 times, and positive blood serum of mouse that deliquated 1:128 times. We used the test paper strip, RBPT and SAT to detections 45 mouse serum immuned by Brucella M5, the positive rate are, respectively,95.6%(43/45),86.7%(39/45),93.3%(42/45). The coincidence of test paper strip and RBPT was 90.6%, the coincidence of test paper strip and SAT was 97.9%. The test paper strip could conserve six months in 4℃.Conclusion the test paper strip can be used in the quick diagnosis of Brucella antibody.To establish a method of duplex real-time fluorescence quantitative PCR assay detect Brucella and Mycobacterium tuberculosis simultaneously. We screened the optimimally specific primers of Brucella combined with the spesific cfp10 primers of Mycobacterium tuberculosis to establish this method. We detected the specificity, sensitivity and reproducibility of this method and detected the clinic and imitation samples. The results showed that the cmp25 primers of Brucella combine with cfp10 of Mycobacterium tuberculosis was the optimization.The Tm of duplex RT-PCR to amplify Brucella and Mycobacterium tuberculosis was 88℃-89℃and 90℃-91℃, But the result of amplification of other bacterias were negative. The lowest detection limit for DNA of Brucella, Mycobacterium tuberculosis or Brucella and Mycobacterium tuberculosis was 20copies/ul, 50copies/ul,100copies/ul, respectively, Higher 100 times than conventional PCR. Use the assay and conventional PCR to detect clinical sample and analogic sample, the coincidence was 100%. The assay could detected Brucella and Mycobacterium tuberculosis simultaneously. the assay have satisfactory specificity, sensitivity and reproducibility.