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Low-stringency single-specific-primer PCR as a tool for detection of mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis.

ABSTRACT: By the low-stringency single-specific-primer PCR technique, a highly sensitive and rapid method for diagnosis of rifampin resistance in Mycobacterium tuberculosis was established. Seven rifampin-resistant and five rifampin-susceptible specimens were analyzed. Rifampin resistance was determined by MIC measurement. A complex electrophoretic pattern consisting of many bands was obtained for both susceptible and rifampin-resistant isolates. The same pattern was obtained for all of the susceptible specimens, but differences between resistant and susceptible isolates were found. DNA sequencing showed that a particular mutation produces a specific electrophoretic pattern.

SUBMITTER: Carvalho WS

PROVIDER: S-EPMC165332 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Low-stringency single-specific-primer PCR as a tool for detection of mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis.

Carvalho Wania S WS Spindola de Miranda Silvana S Costa Kátia M KM Araújo José G V C JG Augusto Claudio J CJ Pesquero João B JB Pesquero Jorge L JL Gomes Maria A MA

Journal of clinical microbiology 20030701 7

By the low-stringency single-specific-primer PCR technique, a highly sensitive and rapid method for diagnosis of rifampin resistance in Mycobacterium tuberculosis was established. Seven rifampin-resistant and five rifampin-susceptible specimens were analyzed. Rifampin resistance was determined by MIC measurement. A complex electrophoretic pattern consisting of many bands was obtained for both susceptible and rifampin-resistant isolates. The same pattern was obtained for all of the susceptible sp ...[more]

PMID: 12843099

Similar Datasets

Rapid detection of rpoB gene mutations conferring rifampin resistance in Mycobacterium tuberculosis.

Project description:Multidrug-resistant Mycobacterium tuberculosis strains are widespread and present a challenge to effective treatment of this infection. The need for a low-cost and rapid detection method for clinically relevant mutations in Mycobacterium tuberculosis that confer multidrug resistance is urgent, particularly for developing countries. We report here a novel test that detects the majority of clinically relevant mutations in the beta subunit of the RNA polymerase (rpoB) gene that confer resistance to rifampin (RIF), the treatment of choice for tuberculosis (TB). The test, termed TB ID/R, combines a novel target and temperature-dependent RNase H2-mediated cleavage of blocked DNA primers to initiate isothermal helicase-dependent amplification of a rpoB gene target sequence. Amplified products are detected by probes arrayed on a modified silicon chip that permits visible detection of both RIF-sensitive and RIF-resistant strains of M. tuberculosis. DNA templates of clinically relevant single-nucleotide mutations in the rpoB gene were created to validate the performance of the TB ID/R test. Except for one rare mutation, all mutations were unambiguously detected. Additionally, 11 RIF-sensitive and 25 RIF-resistant clinical isolates were tested by the TB ID/R test, and 35/36 samples were classified correctly (96.2%). This test is being configured in a low-cost test platform to provide rapid diagnosis and drug susceptibility information for TB in the point-of-care setting in the developing world, where the need is acute.

| S-EPMC3405577 | biostudies-literature

Frequency of rpoB mutations inside and outside the cluster I region in rifampin-resistant clinical Mycobacterium tuberculosis isolates.

Project description:The prevalence of recently described mutation V176F, located in the beginning of the rpoB gene and associated with rifampin resistance and the wild-type cluster I sequence, was determined by analyzing the distribution of rpoB mutations among 80 rifampin (RIF)-resistant Mycobacterium tuberculosis strains isolated in Germany during 1997. The most frequent rpoB mutations were changes in codon 456 (52 isolates, 65%), followed by changes in codon 441 (13 isolates, 16%) and codon 451 (11 isolates, 14%). The V176F mutation was detected in one isolate of the study population and in 5 of 18 RIF-resistant strains with no cluster I mutation from six previously published studies. In three isolates, a mixture of resistant and susceptible subpopulations (heteroresistance) prohibited the detection of rpoB mutations in the initial analysis; however, in these isolates, cluster I mutations could be verified after a passage on RIF-containing medium. IS6110 DNA fingerprinting of 76 strains revealed eight clusters comprising 27 strains with identical restriction fragment length polymorphism patterns that mainly also show identical rpoB mutations and identical or similar drug resistance patterns. In conclusion, our results indicate that the V176F mutation should be included in molecular tests for prediction of RIF resistance in M. tuberculosis. We further demonstrated that heteroresistance caused by a mixture of mycobacterial subpopulations with different susceptibilities to RIF may influence the sensitivity of molecular tests for detection of resistance.

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Profiling of rpoB mutations and MICs for rifampin and rifabutin in Mycobacterium tuberculosis.

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| S-EPMC4042728 | biostudies-literature

Visual detection of rpoB mutations in rifampin-resistant Mycobacterium tuberculosis strains by use of an asymmetrically split peroxidase DNAzyme.

Project description:Multidrug-resistant Mycobacterium tuberculosis is resistant to two first-line antituberculosis drugs, isoniazid and rifampin, resulting in the relapse of tuberculosis. M. tuberculosis grows very slowly, and thus traditional examination methods take time to test its drug resistance and cannot meet clinical needs. The use of a DNA probe makes it possible to test rifampin resistance. We developed an asymmetrical split-assembly DNA peroxidase assay to detect drug-resistant mutation of rifampin-resistant M. tuberculosis in the rpoB gene rapidly and visibly. A new strategy was also designed to eliminate the adverse effects caused by the complicated secondary structure of the target DNA and to improve the efficiency of the probes. This detection system consists of five group detections, covers rifampin-resistant determination region of the rpoB gene, and tests 40 kinds of mutations, including the most common mutations at codons 531 and 526. Every group detection or individual mutant allele detection can distinguish corresponding mutant DNA sequences from the wild-type DNA sequences.

| S-EPMC3486259 | biostudies-literature

rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates from rural areas of Zhejiang, China.

Project description:ObjectiveThe aim was to analyze genetic mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis isolates (RIFR-MTB) from Zhejiang, China.MethodsWe prospectively analyzed RIFR-associated mutations in 13 rural areas of Zhejiang. Isolates were subjected to species identification, phenotype drug susceptibility testing (DST), DNA extraction, and rpoB gene sequencing.ResultsA total of 103 RIFR isolates were identified by DST (22 RIFR only, 14 poly-drug resistant, 49 multidrug resistant, 13 pre-extensively drug resistant [pre-XDR], and 5 extensively drug resistant [XDR]) from 2152 culture-positive sputum specimens. Gene sequencing of rpoB showed that the most frequent mutation was S450L (37.86%, 39/103); mutations P280L, E521K, and D595Y were outside the rifampicin resistance-determining region (RRDR) but may be associated with RIFR. Mutations associated with poly-drug resistant, pre-XDR, and XDR TB were mainly located at codon 445 or 450 in the RRDR.ConclusionsThe frequency of rpoB RRDR mutation in Zhejiang is high. Further studies are needed to clarify the relationships between RIFR and the TTC insertion at codon 433 in the RRDR and the P280L and D595Y mutations outside the RRDR.

| S-EPMC7952843 | biostudies-literature

Upregulation of the phthiocerol dimycocerosate biosynthetic pathway by rifampin-resistant, rpoB mutant Mycobacterium tuberculosis.

Project description:Multidrug-resistant tuberculosis has emerged as a major threat to tuberculosis control. Phylogenetically related rifampin-resistant actinomycetes with mutations mapping to clinically dominant Mycobacterium tuberculosis mutations in the rpoB gene show upregulation of gene networks encoding secondary metabolites. We compared the expressed proteomes and metabolomes of two fully drug-susceptible clinical strains of M. tuberculosis (wild type) to those of their respective rifampin-resistant, rpoB mutant progeny strains with confirmed rifampin monoresistance following antitubercular therapy. Each of these strains was also used to infect gamma interferon- and lipopolysaccharide-activated murine J774A.1 macrophages to analyze transcriptional responses in a physiologically relevant model. Both rpoB mutants showed significant upregulation of the polyketide synthase genes ppsA-ppsE and drrA, which constitute an operon encoding multifunctional enzymes involved in the biosynthesis of phthiocerol dimycocerosate and other lipids in M. tuberculosis, but also of various secondary metabolites in related organisms, including antibiotics, such as erythromycin and rifamycins. ppsA (Rv2931), ppsB (Rv2932), and ppsC (Rv2933) were also found to be upregulated more than 10-fold in the Beijing rpoB mutant strain relative to its wild-type parent strain during infection of activated murine macrophages. In addition, metabolomics identified precursors of phthiocerol dimycocerosate, but not the intact molecule itself, in greater abundance in both rpoB mutant isolates. These data suggest that rpoB mutation in M. tuberculosis may trigger compensatory transcriptional changes in secondary metabolism genes analogous to those observed in related actinobacteria. These findings may assist in developing novel methods to diagnose and treat drug-resistant M. tuberculosis infections.

| S-EPMC3497527 | biostudies-literature

rpoB Mutations and Effects on Rifampin Resistance in Mycobacterium tuberculosis.

Project description:ObjectiveTo investigate the mutations within the whole rpoB gene of Mycobacterium tuberculosis and analyze their effects on rifampin (RIF) resistance based on crystal structure.MethodsWe sequenced the entire rpoB gene in 175 tuberculosis isolates and quantified their minimum inhibitory concentrations using microplate-based assays. Additionally, the structural interactions between wild-type/mutant RpoB and RIF were also analyzed.ResultsResults revealed that a total of 34 mutations distributed across 17 different sites within the whole rpoB gene were identified. Of the 34 mutations, 25 could alter the structural interaction between RpoB and RIF and contribute to RIF resistance. Statistical analysis showed that S450L, H445D, H445Y and H445R mutations were associated with high-level RIF resistance, while D435V was associated with moderate-level RIF resistance.ConclusionSome mutations within the rpoB gene could affect the interaction between RpoB and RIF and thus are associated with RIF resistance. These findings could be helpful to design new antibiotics and develop novel diagnostic tools for drug resistance in TB.

| S-EPMC8502021 | biostudies-literature

Rapid detection of rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates in shanghai by using the amplification refractory mutation system.

Project description:Rapid detection of drug resistance in Mycobacterium tuberculosis is essential for efficient treatment and control of this pathogen. The amplification refractory mutation system (ARMS) was used to detect mutations in the rifampin resistance-determining region of the rpoB gene. A total of 39 rifampin-resistant M. tuberculosis isolates in Shanghai were analyzed by this assay, resulting in 92.3% sensitivity (36 of 39) and 87.2% concordance (34 of 39) relative to DNA sequencing, by which 41 mutations of 11 different types, including 9 point mutations and 2 deletions, were identified in the rpoB gene. The most frequent mutations were those associated with codon 531 (21 of 39 [53.8%]) and codon 526 (9 of 39 [23.1%]). The results suggest that the ARMS assay is rapid and simple to implement and could be performed for detection of rifampin resistance in M. tuberculosis to complement conventional culture-based methods.

| S-EPMC150287 | biostudies-literature

Characterization of rpoB mutations in rifampin-resistant clinical isolates of Mycobacterium tuberculosis from Turkey by DNA sequencing and line probe assay.

Project description:Mutations in an 81-bp region of the rpoB gene associated with rifampin resistance were studied in 41 rifampin-resistant clinical strains of Mycobacterium tuberculosis isolated in Turkey. Fourteen different rpoB alleles, three of which had not been reported before, were found. A reverse hybridization-based line probe assay (the Inno-LiPA Rif.TB test) for rapid detection of the mutations was evaluated with these isolates. Rifampin resistance was correctly identified in 23 of 41 isolates (56.1%) with the kit's R probes specific for these mutations. Seventeen of 41 isolates (41.5%) yielded hybridization patterns, with at least one negative signal obtained with the S probes for the wild type. One isolate was identified as rifampin sensitive by the line probe assay. The rate of concordance of the results of the line probe assay with the results of the in vitro susceptibility test was high (97.6%). These results demonstrate that the line probe assay kit may be useful for the rapid diagnosis of rifampin-resistant tuberculosis.

| S-EPMC154651 | biostudies-literature

Rifampin resistance in Mycobacterium kansasii is associated with rpoB mutations.

Project description:Rifampin is the most potent drug used in the treatment of disease due to Mycobacterium kansasii. A 69-bp fragment of rpoB, the gene that encodes the beta subunit of the bacterial RNA polymerase, was sequenced and found to be identical in five rifampin-susceptible clinical isolates of M. kansasii. This sequence showed 87% homology with the Mycobacterium tuberculosis gene, with an identical deduced amino acid sequence. In contrast, missense mutations were detected in the same fragment amplified from five rifampin-resistant isolates. A rifampin-resistant strain generated in vitro also harbored an rpoB gene missense mutation that was not present in the parent isolate. All mutations detected (in codons 513, 526, and 531) have previously been described in rifampin-resistant M. tuberculosis isolates. Rifampin MICs determined by E-test were <1 mg/liter for all rifampin-susceptible isolates and >256 mg/liter for all rifampin-resistant ones. In addition, four of the five rifampin-resistant isolates were also resistant to rifabutin. We have thus shown a strong association between rpoB gene missense mutations and rifampin resistance in M. kansasii. Although our results are derived from a small number of isolates and confirmation with larger numbers would be useful, they strongly suggest that mutations within rpoB form the molecular basis of rifampin resistance in this species.

| S-EPMC90781 | biostudies-literature

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