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.a grant from The National S&T Major Special Project on Major New Drug Innovation (2012ZX09301002-005-003) from the Ministry of Science and Technology of Chin
