Rapid, High-Throughput Detection of Rifampin Resistance and Heteroresistance in Mycobacterium tuberculosis by Use of Sloppy Molecular Beacon Melting Temperature Coding
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.
Pharmacology, Toxicology and Pharmaceutical Science
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Other
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Unknown
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Attention Score in Context
Attention Score in Context
This research output has an Altmetric Attention Score of 16. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 13 January 2026.
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#2,874,055
of 33,695,692 outputs
Outputs from Journal of Clinical Microbiology
#1,371
of 17,438 outputs
Outputs of similar age
#15,727
of 209,090 outputs
Outputs of similar age from Journal of Clinical Microbiology
#9
of 109 outputs
Altmetric has tracked 33,695,692 research outputs across all sources so far. Compared to these this one has done particularly well and is in the 91st percentile: it's in the top 10% of all research outputs ever tracked by Altmetric.
So far Altmetric has tracked 17,438 research outputs from this source. They typically receive more attention than average, with a mean Attention Score of 8.6. This one has done particularly well, scoring higher than 92% of its peers.
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We're also able to compare this research output to 109 others from the same source and published within six weeks on either side of this one. This one has done particularly well, scoring higher than 91% of its contemporaries.
