关键词: 布氏杆菌, 结核杆菌, 二重实时荧光定量PCR

Abstract: To establish a method of duplex real-time fluorescence quantitative PCR assay for detecting Brucella and Mycobacterium tuberculosis simultaneously, we screened the optimimally specific primers of Brucella combined with the spesific cfp10 primers of Mycobacterium tuberculosi. Then the method was constructed and optimized. The specificity, sensitivity and reproducibility of this method were tested, and the clinic and imitation samples were detected. The results showed that the combination of omp25 primers of Brucella with cfp10 of Mycobacterium tuberculosis was optimun. The Tm of duplex RT-PCR to amplify Brucella and Mycobacterium tuberculosis was 88-89 ℃ and 90-91℃, and 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 20, 50 and 100 copies·μL^-1, respectively, 100 times higher than conventional PCR. When the clinical and analogic samples were evaluated in parallel by this new assay and conventional PCR, the coincidental rate between the two tests was 100%. These results show that the assay is specific, sensitive and repetitive, and could be used in detecting Brucella and Mycobacterium tuberculosis simultaneously.

Key words: Brucella, Mycobacterium tuberculosis, duplex realtime fluorescence quantitative PCR