Establishment of the denaturing high-performance liquid chromatography combined with multiplex nucleic acid amplification method for rapid identification of four important pathogenic mycobacteria / 中国人兽共患病学报

ABSTRACT

A new molecular method for simultaneously rapid detection and differentiation of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium and Mycobacterium paratuberculosis was established by using denaturing high-performance liquid chromatography (DHPLC) combined with multiplex nucleic acid amplification. These 4 important pathogenic mycobacteria were identified by separation of 4 specific PCR-amplified target fragments by DHPLC analysis. A total of 51 Mycobacterium strains and 22 other bacterial species were tested to confirm the specificity of the multiplex PCR-DHPLC assay. The sensitivity of the assay was as low as 10~2-10~3 gene copies. This method rapidly identify the positive clinical samples from human and bovine with higher detection ratio than traditional culture method and was able to identify simultaneously four pathogenic Mycobacterium, which provided a new molecular tool for rapid detection of tuberculosis and paratuberculosis in human and animals.