ArticleJournal of clinical microbiology2000
Use of real-time PCR and fluorimetry for rapid detection of rifampin and isoniazid resistance-associated mutations in Mycobacterium tuberculosis.
Article in Journal of clinical microbiology, 2000. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 39 papers.
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39 citing papers in PubMed, 129 citations in OpenAlex.
- Rapid Extensively Drug-Resistant (XDR) TB Diagnosis: An In-House DNA Biochip for Drug Resistance Detection and Mutation Profiling ofThe Canadian journal of infectious diseases & medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie medicale · 2026Article
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- Rapid Detection of Rifampicin- and Isoniazid-ResistantJundishapur journal of microbiology · 2016Article
- Methodological and Clinical Aspects of the Molecular Epidemiology of Mycobacterium tuberculosis and Other Mycobacteria.Clinical microbiology reviews · 2016Review
- Article
- Visual detection of rpoB mutations in rifampin-resistant Mycobacterium tuberculosis strains by use of an asymmetrically split peroxidase DNAzyme.Journal of clinical microbiology · 2012Article
- Fast test for assessing the susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin by real-time PCR.Memorias do Instituto Oswaldo Cruz · 2012Article
- Rapid diagnosis of extensively drug-resistant tuberculosis by use of a reverse line blot hybridization assay.Journal of clinical microbiology · 2011Article
- Laboratory diagnosis of tuberculosis in resource-poor countries: challenges and opportunities.Clinical microbiology reviews · 2011Review
- Multi-probe real-time PCR identification of common Mycobacterium species in blood culture broth.The Journal of molecular diagnostics : JMD · 2009Article
- Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes.Current genomics · 2007Article
- Real-time PCR in clinical microbiology: applications for routine laboratory testing.Clinical microbiology reviews · 2006Review
- Rapid determination of rifampin resistance in clinical isolates of Mycobacterium tuberculosis by real-time PCR.Journal of clinical microbiology · 2005Article
- Genetic diversity, determined on the basis of katG463 and gyrA95 polymorphisms, Spoligotyping, and IS6110 typing, of Mycobacterium tuberculosis complex isolates from Italy.Journal of clinical microbiology · 2005Article
- Advances in real-time PCR: application to clinical laboratory diagnostics.Advances in clinical chemistry · 2005Review
- Rapid detection of rifampin resistance in Mycobacterium tuberculosis isolates from India and Mexico by a molecular beacon assay.Journal of clinical microbiology · 2004Article
- Rapid direct detection of multiple rifampin and isoniazid resistance mutations in Mycobacterium tuberculosis in respiratory samples by real-time PCR.Antimicrobial agents and chemotherapy · 2004Article
- Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR.Journal of clinical microbiology · 2004Article
- Peptide nucleic acid-mediated competitive PCR clamping for detection of rifampin-resistant Mycobacterium tuberculosis.Antimicrobial agents and chemotherapy · 2004Article
- Molecular characterization of rifampin- and isoniazid-resistant Mycobacterium tuberculosis strains isolated in Poland.Journal of clinical microbiology · 2004Article
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Very fast amplification of DNA in small volumes can be continuously monitored with a rapid cycler that incorporates fluorimetric detection. Primers were designed to amplify a 157-bp fragment of the rpoB gene spanning codons 526 and 531 and a 209-bp fragment of the katG gene spanning codon 315 of Mycobacterium tuberculosis. Most mutations associated with resistance to rifampin (RMP) and isoniazid (INH) in clinical isolates occur in these codons. Two pairs of hybridization probes were synthesized; one in each pair was 3' labeled with fluorescein and hybridized upstream of the codon with the mutation; the other two probes were 5' labeled with LightCycler-Red 640. Each pair of probes recognized adjacent sequences in the amplicon. After DNA amplification was finished by using a LightCycler, the temperature at which the Red 640 probe melted from the product was determined in a 3-min melt program. Twenty M. tuberculosis clinical isolates susceptible to streptomycin, INH, RMP, and ethambutol and 36 antibiotic-resistant clinical M. tuberculosis isolates (16 resistant to RMP, 16 to INH, and 4 to both antimicrobial agents) were amplified, and the presence of mutations was determined using single-strand conformation polymorphism analysis, the LiQor automated sequencer, and the LightCycler system. Concordant results were obtained in all cases. Within 30 min, the LightCycler method correctly genotyped all the strains without the need of any post-PCR sample manipulation. Overall, this pilot study demonstrated that real-time PCR coupled to fluorescence detection is the fastest available method for the detection of RMP and INH resistance-associated mutations in M. tuberculosis clinical isolates.
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