Rolling circle amplification for direct detection of rpoB gene mutations in Mycobacterium tuberculosis directly from clinical specimens

Abstract

Rapid and accurate detection of multidrug resistance (MDR) inMycobacterium tuberculosisis essential to improve treatment outcomes and reduce global transmission but remains a challenge. Rifampin (RIF) resistance is a reliable marker of MDR tuberculosis (TB) since by far the majority of RIF-resistant strains are also isoniazid (INH) resistant. We have developed a rapid, sensitive, and specific method for detecting the most common mutations associated with RIF resistance, in the RIF resistance determining region (RRDR) ofrpoB, using a cocktail of six padlock probes and rolling circle amplification (RCA). We used this method to test 46 storedM. tuberculosisclinical isolates with known RIF susceptibility profiles (18 RIF resistant, 28 susceptible), a standard susceptible strain (H37Rv, ATCC 27294) and 78M. tuberculosisculture-positive clinical (sputum) samples, 59 of which grew RIF-resistant strains. All stored clinical isolates were correctly categorized, by the padlock probe/RCA method, as RIF susceptible or resistant; the sensitivity and specificity of the method, for direct detection of phenotypically RIF-resistantM. tuberculosisin clinical specimens, were 96.6 and 89.5%, respectively. This method is rapid, simple, and inexpensive and has the potential for high-throughput routine screening of clinical specimens for MDRM. tuberculosis, particularly in high prevalence settings with limited resources.