Abstract
Rifampin resistance in
Mycobacterium tuberculosis
is largely determined by mutations in an 80-bp rifampin resistance determining region (RRDR) of the
rpoB
gene. We developed a rapid single-well PCR assay to identify RRDR mutations. The assay uses sloppy molecular beacons to probe an asymmetric PCR of the
M. tuberculosis
RRDR by melting temperature (
T
m
) analysis. A three-point
T
m
code is generated which distinguishes wild-type from mutant RRDR DNA sequences in approximately 2 h. The assay was validated on synthetic oligonucleotide targets containing the 44 most common RRDR mutations. It was then tested on a panel of DNA extracted from 589 geographically diverse clinical
M. tuberculosis
cultures, including isolates with wild-type RRDR sequences and 25 different RRDR mutations. The assay detected 236/236 RRDR mutant sequences as mutant (sensitivity, 100%; 95% confidence interval [CI], 98 to 100%) and 353/353 RRDR wild-type sequences as wild type (specificity, 100%; 95% CI, 98.7 to 100%). The assay identified 222/225 rifampin-resistant isolates as rifampin resistant (sensitivity, 98.7%; 95% CI, 95.8 to 99.6%) and 335/336 rifampin-susceptible isolates as rifampin susceptible (specificity, 99.7%; 95% CI, 95.8 to 99.6%). All mutations were either individually identified or clustered into small mutation groups using the triple
T
m
code. The assay accurately identified mixed (heteroresistant) samples and was shown analytically to detect RRDR mutations when present in at least 40% of the total
M. tuberculosis
DNA. This was at least as accurate as Sanger DNA sequencing. The assay was easy to use and well suited for high-throughput applications. This new sloppy molecular beacon assay should greatly simplify rifampin resistance testing in clinical laboratories.