Novel rapid PCR for the detection of Ile491Phe rpoB mutation of Mycobacterium tuberculosis, a rifampicin-resistance-conferring mutation undetected by commercial assays

Abstract

ObjectivesNeither the liquid medium-based Bactec MGIT, nor commercial molecular assays such as the Xpert MTB/RIF and the MTBDRplus V2.0 assays are capable of detecting up to 30% of rifampicin-resistant Mycobacterium tuberculosis strains in Swaziland because of the large proportion of the rpoB Ile491Phe mutations. In other countries, the frequency of this mutation is thought to be low. MethodsWe designed a real-time multiplex allele-specific PCR assay to identify the rpoB Ile491Phe mutation responsible for these undetected resistant M. tuberculosis strains. ResultsThe technique showed 100% similarity with rpoB sequencing on a panel of 78 strains from Swaziland. ConclusionsWe propose that the detection of the rpoB Ile491Phe rpoB mutation should complement commercial assays for the diagnosis of rifampicin-resistant M. tuberculosis in routine conditions, particularly in countries where this specific mutation is frequent. The technique proposed in this paper is adapted for most reference laboratories.