Rapid Detection of Mycobacterium Tuberculosis Complex in Sputum and in Culture Growth by LAMP Test

Abstract

       Loop-mediated isothermal amplification (LAMP) is a new nucleic acid amplification assay, which could be conducted under isothermal conditions (65oC). The reaction could be completed within 2 hours without the need of PCR machine and the amplicons could be detected by naked eye visualization. Because of the time-consuming in culturing Mycobacterium tuberculosis due to its slow growth rate, the development and application of LAMP for rapid diagnosis of tuberculosis would be useful. In this study, an efficient LAMP which specifically detected members of Mycobacterium tuberculosis complex was developed. Species-specific primers were designed by targeting 16S rRNA gene of M. tuberculosis. The method was sensitive, as it could detect as low as nine
bacilli in decontaminated sputum. When it was used in direct detection of M. tuberculosis in sputum, LAMP method showed positive results in 73 of 73 smear positive-culture positive tuberculosis specimens (100%) and in 15 of 20 smear negative-culture positive tuberculosis specimens (75%). In addition, LAMP was evaluated for rapid identification of culture isolates grown in a liquid medium
(MGIT). Ninety nine sputum specimens from 135 patients with clinically diagnosed pulmonary tuberculosis were positive according to the Bactec MGIT 960 system. Among these, 96 were positive and 3 were negative according to the LAMP test. Compared to the convention identification methods by biochemical tests; Niacin, Nitrate reduction and Catalase tests (at 68oC, pH 7),
LAMP method correctly identified all 96 M. tuberculosis isolates and gave negative results for all non-tuberculous mycobacteria. LAMP method was also able to rapidly identify M. tuberculosis grown on solid media. In conclusion, the fast, easy, sensitive, and specific LAMP test established here might be useful for rapid detection and identification of M. tuberculosis in clinical specimens.
Key words: tuberculosis, Mycobacterium tuberculosis, LAMP, MGIT