Single strand conformation polymorphism profiles with biotinylated PCR products to detect mutations in rpoB gene of Mycobacterium tuberculosis

摘要

A fragment of the rpoB gene, including the regionudshown to be involved in rifampicin resistance, wasudamplified from 15 rifampicin-resistant and 6 rifampicin-udsensitive clinical isolates of Mycobacteriumudtuberculosis by the polymerase chain reaction (PCR).udOne of the primers, employed in PCR, was biotinylated.udThe biotinylated strand of the PCR productudwas separated from the unbiotinylated strand usingudstreptavidin magnetic beads. Both the strands wereudsubjected to single strand conformation polymorphismudin polyacrylamide gel electrophoresis. The DNA bandsudwere silver stained to study their migration pattern.udA shift in the migration of either of the strands ofudthe test strain compared to the strands from a controludrifampicin-sensitive strain was considered as indicativeudof resistance. This strategy was found to ease theudvisualization of shift in the migration of the strandsudin 17 of 21 samples and thereby detection of mutations.