PCR-SSCP method to detect genetic mutations in rpoB gene as a marker of rifampi cin-resistance was developed by Telenti et al., and we have modified it applying nonradioactive PhastSystem for more practical use in the detection of rifampicin-resistance of Mycobacterium tuberculosis.
PCR products amplified with the primers specific to rpoB gene using extracted DNA from 89 strains of M. tuberculosis were sequenced and the amino acid sequences were morphism was determined by the PhastSystem. The bands were stained by silver staining.
Among 89 strains of M. tuberculosis, 43 were confirmed as rifampicin-resistant (RFPr) and 46 were rifampicin-sensitive (RFPs) by the culture on the drug-containing media. All of the 43 RFPr strains had one or more mutations in the DNA sequence of rpoB gene, while none of the RFPs strains had such mutations. However, by PCR-SSCP, only 20 out of 43 RFPr strains showed clear differences in the band pattern of electrophoresis from that of RFPs strains. Other 23 RFPr strains had only slight differences in the band pattern of the PCR-SSCP from that of RFPs strains. But it was noticed that the main bands of RFPr strains were distinguishable from the main bands of RFPs strains even their patterns were similar. Thus, it is possible to apply a non-radioactive PCR-SSCP for the detection of rifampicin resistance of M. tuberculosis with further improvement of the condition of gelelectrophoresis or staining techniques.
