Rapid detection of Rifampicin-resistant M. tuberculosisby PCR-SSCP of rpoB gene

Shim, Tae Sun; Yoo, Chul Gyu; Han, Sung Koo; Shim, Young Soo; Kim, Young Whan

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) M. tuberculosis. rpoB gene encodes the beta-subunit of RNA polymerase of M. tuberculosis which is the target of RFP. Recent reports show that rpoB gene mutations are the cause of RFP resistance of M. tuberculosis and the main mechanism of rpoB gene mutation is point mutation. And PCR-SSCP is a rapid and easy method for detecting point mutations. So we performed PCR-SSCP of rpoB gene of M. tuberculosis and compared the result with traditional RFP sensitivity test. Method: The 27 RFP sensitive M. tuberculosis culture isolates and 25 RFP resistant isolates were evaluated. The RFP sensitivity test was done at the Korean Tuberculosis Istitute. The DNA was extracted by bead beater method and was amplified with primers TR-8 and TR-9 in a 20ul PCR reaction containing O.1ul(luCi) [alpha - 32p] -dCTP. After amplification, SSCP was done using non-denaturating polyacrylamide gel electrophoresis. Then direct sequencing was done in cases of different eletrophoretic mobility compared with that of H37Rv. In 19 cases, we compared PCR-SSCP results with patient's clinical course and the results of traditional RFP sensitivity test.
Results
1) All 27 RFP sensitive M. tuberculosis isolates showed the same electrophoretic mobility compared with that of H37Rv. And all 25 RFP resistant M. tuberculosis isolates showed different electrophoretic mobility. 2) The mechanism of rpoB gene mutation of M. tuberculosis is mainly point mutation. 3) The PCR-SSCP results correlate well with traditional RFP sensitivity and patient's clinical response to antituberculous treatment.
Conclusion
The PCR-SSCP of rpoB gene is a very sensitive and rapid mehod in detecting RFP- resistant M. tuberculosis.