MYCOBACTERIUM COMPLEX IDENTIFICATION BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
Abstract
Introduction: There are different ways for identification of Mycobacteria. One of the most sensitive method is HPLC of phenacyl esters of mycolic acids of Mycobacteria for rapid identification of them after their primary cultures. This study uses HPLC for rapid identification and dissociation of Mycobacterium tuberculosis complex.
Methods: In this study we use HPLC patterns of mycolic acids for identification three important species of mycobacteria (M. tuberculosis, M. bovis, M. bovis BCG) from other mycobacterial species. All the strains were obtained from Tuberculosis and Pulmonary Diseases Research Center. HPLC conditions was as follows: HPLC: Model 1200 Cecil, Column: URP C-18 25X4.6 mm, Detector: U.V variable wave length at 254 nm, Elution: Gradient of methanol/chloroform. Flow rate: 2.5 ml/min.
Results: HPLC leads to obtaining chromatograms which on its X-axis retention times (of different peaks which exist in the sample) and on its Y-axis U.V absorbance (of these peaks) were drown. These chromatograms in M. bovis and M. tuberculosis samples are similar with each other but differs from BCG ones.
Discussion: On the basis of different retention times and numbers of the peaks which present in each chromatogram, we can differentiate between M. bovis, M. tuberculosis and BCG from other Mycobacteria. Also, with this method we can identify BCG from M. bovis and M. tuberculosis (because BCG has 9 and M. bovis and M. tuberculosis has 7 characteristic peaks in their chromatograms).
Methods: In this study we use HPLC patterns of mycolic acids for identification three important species of mycobacteria (M. tuberculosis, M. bovis, M. bovis BCG) from other mycobacterial species. All the strains were obtained from Tuberculosis and Pulmonary Diseases Research Center. HPLC conditions was as follows: HPLC: Model 1200 Cecil, Column: URP C-18 25X4.6 mm, Detector: U.V variable wave length at 254 nm, Elution: Gradient of methanol/chloroform. Flow rate: 2.5 ml/min.
Results: HPLC leads to obtaining chromatograms which on its X-axis retention times (of different peaks which exist in the sample) and on its Y-axis U.V absorbance (of these peaks) were drown. These chromatograms in M. bovis and M. tuberculosis samples are similar with each other but differs from BCG ones.
Discussion: On the basis of different retention times and numbers of the peaks which present in each chromatogram, we can differentiate between M. bovis, M. tuberculosis and BCG from other Mycobacteria. Also, with this method we can identify BCG from M. bovis and M. tuberculosis (because BCG has 9 and M. bovis and M. tuberculosis has 7 characteristic peaks in their chromatograms).
Keywords
M. tuberculosis, M bovis, BCG; p-bromophenacyl bromide, HPLC