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To establish a method for detection of M. tuberculosis by molecular beacon real-time PCR and evaluate its application in the clinical detection of M. tuberculosis.The primers and molecular probe were designed to M. tuberculosis specific DNA sequence for Ag85B, and constitute a standards. 158 sputum specimens from patients with pulmonary tuberculosis were detected using molecular beacon real-time PCR, acid-fast stain and cultivation, and the detection rates of the three methods were compared.The lowest detection limit of the detected method is 2 genome copies per reaction, the linear standard curve was obtained between 2 x 10(2) and 2 x 10(8) DNA copies/mL. The method detected 5 strains of non-mycobacterium, 6 strains of non-tuberculosis mycobacterium and M. tuberculosis H37Rv, only M. tuberculosis H37Rv had 75 bp specific band. The positive rates of detection using molecular beacon real-time PCR, acid-fast stain and cultivation were 60.13%, 16.46%, 31.01% respectively, the positive rates of detection of real-time PCR was significantly higher than that of acid-fast stain and cultivation.Molecular beacon real-time PCR has high sensitivity and specificity in the detection of M. tuberculosis, which is clinical significance for the diagnosis of tuberculosis.