Abstract:  Based on multiplex PCR combined with denaturing high-performance liquid chromatography technique, this study was carried out for simultaneous detection of Mycobacterium tuberculosis complex and identification of major pathogenic species for human and animal tuberculosis. A novel multiplex PCR-DHPLC method was established which identified four gene targets in an reaction，including IS6110 and IS1081 insertion sequence specific for Mycobacterium tuberculosis complex and specific structure gene for M. tuberculosis and M. bovis respectively. The specification of the quadruplex PCR-DHPLC assay was verified by tests on 13 species of mycobacteria reference and isolated strains and 23 species of other microorganism. The limit of detection was measured by testing on purified mycobacterium DNA and cloned plasmid DNA. Clinical performance of the assay was demonstrated by detection on bovine tissue samples from infected herds and compared with culture. Results were as follows: The assay specifically detected strains of Mycobacterium tuberculosis complex and simultaneously differentiated M. tuberculosis and M. bovis strains. The limit of detection on cloned plasmid DNA was 10² to 10³ gene copy or 3.6 to 6.8 fg DNA per reaction. For the detection on 39 suspected tissue samples, the assay detected 33 samples positive for Mycobacterium tuberculosis complex and identified 28 as M. bovis positive, while 21 samples were showed positive by culture method. The whole detection procedure of the multiplex PCR-DHPLC assay on clinical samples could finish within 1 day, and the detection on purified DNA sample took around 2 hours. This study provided a novel rapid molecular method for differential identification of pathogenic mycobacteria for human and animal tuberculosis.