Direct, automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis

Abstract

A rapid screening test was recently established for the detection of mutations in the rpoB gene of Mycobacterium tuberculosis, a region identified as the locus for rifampin resistance (Rifr). The detection method involved the amplification by polymerase chain reaction (PCR) of the Rifr region and the identification of mutations by single-strand DNA conformation polymorphism analysis (SSCP) of the amplification products. Experience using two different PCR-SSCP formats for the evaluation of BACTEC cultures and sputum is presented here; the previously described manual procedure for the detection for the detection of radiolabelled amplification products and an automated SSCP by which fluorescein-labelled products were detected on a Pharmacia DNA sequencer apparatus. All 17 different Rifr mutations known to date were consistently detected. PCR-SSCP could be used for the evaluation of minimally grown cultures (BACTEC 12B medium with a growth index of < or = 100) and for direct screening of microscopically positive sputa with greater than 10 organisms per field (magnification, x250). Implementation of this technique could result in rapid detection of rifampin resistance in M. tuberculosis, a marker of multidrug-resistant tuberculosis.