Detection of Pyrazinamide-Resistant Mycobacteriumtuberculosis by PCR-SSCP of pncA Gene

Shim, Tae Sun; Kim, Young Whan; Chin, Jae Yong; Lim, Chae Man; Lee, Sang Do; Koh, Younsuck; Kim, Woo Sung; Kim, Dong Soon; Kim, Won Dong

Abstract

BACKGROUND: Recently the incidence of tuberculosis is increasing in many countries and control of the disease is further threatened by the emergence of multi-drug resistant tuberculosis. So rapid detection of drug resistance is very important. Pyrazinamide (PZA) is a first-line chemotherapeutic agent for tuberculosis. Now in Korea, we perform PAase activity test instead of actual pyrazinamide susceptibility test for the detection of PZA resistant M. Tuberculosis. Recently the pncA gene, encoding the PZase of M. Tuberculosis, was completely sequenced. And it was reported that the mutation of pncA gene would be associated with PZA resistance of M. Tuberculosis. Therefore we performed this study to evaluate the possibility for the rapid detection of PZA resistant M. Tuberculosis using RCR-SSCR of pncA gene. METHOD: 44 cultured clinical isolates of M. tuberculosis, BCG Tokyo strain, BCG french strain, and one M.bovis isolate were studied. We used H37Rv as the reference strain, The PZase activity test was done at the reference laboratory of Korean Tuberculosis Institute. DNA was extracted by bead-beater method and 561 bp fragment including pncA gene was amplified by PCR. The PCR product were digested by BstB I enzyme. SSCP was done using MDE gel. Of the 44 strains of M. tuberculosis, 22 strains were PZase-positive and other 22 strains were PZase negative.
RESULTS
Of the 22 PZase positive strains, 18 strains(82%) showed the same mobility compared with that of H37Rv and 4(18%) showed different mobility. Of the PZase negative strains, 19(86%) strains showed the same mobility pattern compared with that H37Rv and 3(14%) showed different mobility. Naturally PZA-resistant BCG-French strain, BCG-Tokyo strain, and one M. bovis isolate showed the same band pattern each other, but their mobility were different from that of H37Rv. The results of PZase activity test and PCR-SSCP of pncA of M tuberculosis were statistically significantly correlated each other(p<0.01).
CONCLUSION
The PCR-SSCP after BstB I restriction of pncA gene of M tuberculosis may be a useful method for the rapid detection of PZA-resistant M. tuberculosis.