Detection of rpoB Gene Mutations Using Helicase-Dependent Amplification

For patients infected with tuberculosis, detection of rpoB gene mutations is critical for diagnosing drug-resistant strains of the causative pathogen, Mycobacterium tuberculosis (MTB). Traditional approaches to drug resistance include culture, which is very slow. Recently described real-time polymerase chain reaction approaches have addressed turnaround time but at relatively high cost. Here, we describe a novel amplification method, termed blocked-primer helicase-dependent amplification, for amplifying rpoB gene sequences in MTB. Resultant amplicon is hybridized to a probe set arrayed on a modified silicon-based chip to determine if there is any mutation in that region. Using this method, we could detect the majority of clinically relevant mutations in rpoB gene.