DIRECT, AUTOMATED DETECTION OF RIFAMPIN-RESISTANT MYCOBACTERIUM-TUBERCULOSIS BY POLYMERASE CHAIN-REACTION AND SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS

A rapid screening test was recently established for the detection of m utations in the rpoB gene of Mycobacterium tuberculosis, a region iden tified as the locus for rifampin resistance (Rif(r)). The detection me thod involved the amplification by polymerase chain reaction (PCR) of the Rif(r) region and the identification of mutations by single-strand DNA conformation polymorphism analysis (SSCP) of the amplification pr oducts. Experience using two different PCR-SSCP formats for the evalua tion of BACTEC cultures and sputum is presented here: the previously d escribed manual procedure for the detection of radiolabelled amplifica tion products and an automated SSCP by which fluorescein-labelled prod ucts were detected on a Pharmacia DNA sequencer apparatus. All 17 diff erent Rif(r) mutations known to date were consistently detected. PCR-S SCP could be used for the evaluation of minimally grown cultures (BACT EC 12B medium with a growth index of less-than-or-equal-to 100) and fo r 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.