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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
Rapid, high-throughput detection of rifampin resistance and heteroresistance in Mycobacterium tuberculosis by use of sloppy molecular beacon melting temperature coding
Journal of Clinical Microbiology, Volume 50, No. 7, Year 2012
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Description
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. Copyright © 2012, American Society for Microbiology. All Rights Reserved.
Available Materials
https://efashare.b-cdn.net/share/pmc/articles/PMC3405605/bin/supp_50_7_2194__index.html
https://efashare.b-cdn.net/share/pmc/articles/PMC3405605/bin/supp_50.7.2194_JCM-JCM00143-12-s01.pdf
https://efashare.b-cdn.net/share/pmc/articles/PMC3405605/bin/supp_50.7.2194_JCM-JCM00143-12-s02.pdf
Authors & Co-Authors
Chakravorty, Soumitesh
United States, Newark
Rutgers new Jersey Medical School
Lee, Jongseok
South Korea, Seoul
International Tuberculosis Research Center
Roh, Sandy S.
United States, Newark
Rutgers new Jersey Medical School
Hwang, Soohee
Unknown Affiliation
Banada, Padmapriya P.
United States, Newark
Rutgers new Jersey Medical School
Safi, Hassan
United States, Newark
Rutgers new Jersey Medical School
Via, L. E.
United States, Bethesda
National Institutes of Health Nih
Cho, Sangnae Ray
South Korea, Seoul
International Tuberculosis Research Center
South Korea, Seoul
Yonsei University
Barry, Clifton Earl
United States, Bethesda
National Institutes of Health Nih
Alland, David
United States, Newark
Rutgers new Jersey Medical School
Statistics
Citations: 37
Authors: 10
Affiliations: 5
Identifiers
Doi:
10.1128/JCM.00143-12
ISSN:
1098660X
Research Areas
Cancer
Genetics And Genomics