中文摘要：

本试验旨在利用二重实时荧光定量PCR技术建立鉴别诊断布氏杆菌和结核杆菌的方法。通过筛选布氏杆菌omp10、omp19、omp17、omp25、omp31和结核杆菌cfp10基因的最佳引物组合,建立二重实时定量PCR鉴别诊断方法,对该方法进行稳定性、特异性和敏感性试验,并对临床样品及模拟样品进行检测。经筛选布氏杆菌的omp25基因与结核杆菌cfp10基因引物组合最佳,该二重实时荧光定量PCR方法中布氏杆菌Tm值为88～89℃,结核杆菌Tm值为90～91℃,对其他供试的菌株则为阴性;该方法对布氏杆菌的DNA最低检出量为20拷贝.μL^-1,结核杆菌为50拷贝.μL^-1,两病原都存在时为100拷贝.μL-1,比常规PCR高100倍。利用所建立的二重荧光定量PCR方法及常规PCR,对24份结核痰样、11份布氏杆菌基因粗提物及30份模拟奶样进行检测,符合率为100%。本试验建立的方法可用于同时检测布氏杆菌和结核杆菌,并且具有良好的特异性、敏感性和稳定性。

英文摘要：

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.