Rapid analysis of resistant mutant genotypes using pyrosequencing.

Abstract

The rapid detection of nucleotide mutations conferring drug resistance is especially important for organisms with long generation times. Mycobacterium tuberculosis, an organism thought to infect around one-third of the global population, is probably the most important of these slow-growers. Multidrug-resistant (MDR) strains of M. tuberculosis, indicated by resistance to rifampicin, are emerging and their rapid detection is crucial for treatment and control of this pathogen. Single nucleotide polymorphisms (SNPs) in the rifampicin resistance determining region (RRDR) of rpoB, an 81-bp region of the RNA polymerase B gene, confer resistance to rifampicin in the vast majority of cases. Combining PCR of this region and rapid short read sequencing using Pyrosequencing enables rapid high-throughput MDR strains to be detected from sputum and early cultures.