Detection of rifampin-resistant Mycobacterium tuberculosis in sputa by nested PCR-linked single-strand conformation polymorphism and DNA sequencing

Either PCR-mediated single strand conformation polymorphism (SSCP) analysis or DNA sequencing of rpoB DNA (157 bp) can be used as a rapid screening me thod for the detection of mutations related to the rifampin resistance of M ycobacterium tuberculosis. However, due to the nonspecific amplification of rpoB DNA from nontuberculous mycobacteria these methods cannot be directly applied to clinical specimens such as sputa. We developed a nested PCR met hod that can specifically amplify the rpoB DNA of M. tuberculosis on the ba sis of rpoB DNA sequences of 44 mycobacteria. Nested PCR-linked SSCP analys is and the DNA sequencing method were applied directly in order to detect M . tuberculosis and determine its rifampin susceptibility in 56 sputa. The r esults obtained by nested PCR-SSCP and DNA sequencing mere concordant with those of conventional drug susceptibility testing and DNA sequencing perfor med with culture isolates.