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.
Key wordsrpoB Mycobacterium tuberculosis Helicase-dependent amplification Gene mutation Mutation detection Drug resistance
We thank Adrianne Clifford and all other members of the Research Group at Great Basin Corporation for technical assistance and Steve Aldous for financial support for this project.
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