Rapid Detection of Rifampicin Resistant M. tuberculosis by PCR-SSCP of rpoB Gene in Clinical Specimens

Shim, Tae Sun; Kim, Young Whan; Lim, Chae Man; Lee, Sang Do; Koh, Youn Suck; Kim, Woo Sung; Kim, Dong Soon; Kim, Won Dong

Abstract

BACKGROUND: Rifampicin(RFP) is a key component of the antituberculous short-course chemotherapy and the RFP resistance is a marker of multi-drug resistant(MDR) tuberculosis. RpoB gene encodes the beta-subunit of RNA polymerase of M. tuberculosis which is the target of RFP. And rpoB gene mutations are the cause of RFP resistance of M. tuberculosis. Although several reports showed that PCR-SSCP would be a rapid diagnostic method for identifying the REP resistance, there were few reports performed using direct, dinical specimens. So we performed PCR-SSCP analysis of rpoB gene of M. tuberculosis in direct, clinical specimens.
METHODS
75 clinical specimens were collected from patients at Asan Medical Center from June to August 1996. After PCR of IS 6110 fragments, 43 both AFB smear-positive and IS 6110 fragment PCR-positive specimens were evaluated. The RFP susceptibility test was referred to the referral laboratory of the Korean Tuberculosis Institute. DNA was extracted by bead beater method. And heminested PCR was done using 0.1ul(1uCi) [alpha-32P]-dCTP. SSCP analysis was done using non-denaturating MDE gel electrophoresis.
RESULTS
The results of PCR of IS 6110 fragments of M. tuberculosis were positive in 55(73%) cases of 75 AFB smear-positive clinical specimens. Of the 55 specimens, RFP susceptibility was confirmed in only 43 specimens. Of the 43 AFB smear-positive and IS6110 fragment-positive specimens, 29 were RFP susceptible and 14 were RFP resistant. All the RFP susceptible 29 strains showed the same mobility compared with that of RFP sensitive H37Rv in SSCP analysis of ropB gene. And all the other RFP resistant 13 strains showed the different mobility. In other words they showed 100% identical results between PCR-SSCP analysis and traditional susceptibility test. CONCLUISON: The PCR-SSCP analysis of rpoB gene in direct clinical specimens could be used as a rapid diagnostic method for detecting RFP resistant M. tuberculosis.