Rapid detection of rifampin resistance in Mycobacterium tuberculosis by Pyrosequencing technology

We developed an assay for rapid detection of rifampin resistance in Mycobacterium tuberculosis based on Pyrosequencing technology, involving a technique for real-time sequencing. A 180-bp region of the rpoB gene was amplified in clinical isolates of both rifampin-resistant and -susceptible M. tuberculosis. The PCR products were subjected to Pyrosequencing analysis using four different sequencing primers in four overlapping reactions. These four sequencing reactions covered the 81-bp region where >96% of the mutations associated with rifampin resistance are located. The results were compared to those obtained with two other molecular methods, the line probe assay and cycle sequencing, and the phenotypic BACTEC method. The genotypic determination methods all detected the mutations that previously have been correlated with rifampin resistance. In addition, Pyrosequencing analysis and the two other molecular methods found additional mutations within the rpoB gene in phenotypically susceptible strains. We found that Pyrosequencing technology, in particular, offers high accuracy, short turnaround time, and a potentially high throughput in detection of rifampin resistance in M. tuberculosis.