title: Rapid, high-throughput detection of rifampin resistance and heteroresistance in Mycobacterium tuberculosis by use of sloppy molecular beacon melting temperature coding.
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.
authors.affiliation: Division of Infectious Disease, Department of Medicine, and the Ruy V. Lourenço Center for Study of Emerging and Reemerging Pathogens, New Jersey Medical School, Newark, New Jersey, USA.
authors.forename: Soumitesh
authors.initials: S
authors.lastname: Chakravorty
authors.forename: Harsheel
authors.initials: H
authors.lastname: Kothari
authors.forename: Bola
authors.initials: B
authors.lastname: Aladegbami
authors.forename: Eun Jin
authors.initials: EJ
authors.lastname: Cho
authors.forename: Jong Seok
authors.initials: JS
authors.lastname: Lee
authors.forename: Sandy S
authors.initials: SS
authors.lastname: Roh
authors.forename: Hyunchul
authors.initials: H
authors.lastname: Kim
authors.forename: Hyungkyung
authors.initials: H
authors.lastname: Kwak
authors.forename: Eun Gae
authors.initials: EG
authors.lastname: Lee
authors.forename: Soo Hee
authors.initials: SH
authors.lastname: Hwang
authors.forename: Padmapriya P
authors.initials: PP
authors.lastname: Banada
authors.forename: Hassan
authors.initials: H
authors.lastname: Safi
authors.forename: Laura E
authors.initials: LE
authors.lastname: Via
authors.forename: Sang-Nae
authors.initials: SN
authors.lastname: Cho
authors.forename: Clifton E
authors.initials: CE
authors.lastname: Barry
authors.forename: David
authors.initials: D
authors.lastname: Alland
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012293:Rifampin / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D013997:Time Factors; D044366:Transition Temperature; D014376:Tuberculosis / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D015345:Oligonucleotide Probes; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin