Abstract
We developed a new approach to DNA sequence analysis that uses fluorogenic reporter molecules—molecular beacons—and demonstrated their ability to discriminate alleles in real-time PCR assays of genomic DNA. A set of overlapping molecular beacons was used to analyze an 81-bp region of the Mycobacterium tuberculosis rpoB gene for mutations that confer resistance to the antibiotic rifampin. In a blinded study of 52 rifampin-resistant and 23 rifampin-susceptible clinical isolates, this method correctly detected mutations in all of the resistant strains and in none of the susceptible strains. The assay was carried out entirely in sealed PCR tubes and was simple to perform and interpret. This approach can be used to analyze any DNA sequence of moderate length with single base pair accuracy.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Tonin, P., Weber, B., Offit, K, Couch, F., Rebbeck, T.R., Neuhausen, S. et al. 1996. Frequency of recurrent BRCA1 and BRCA2 mutations in Ashkenazi Jewish breast cancer families. Nat. Med. 2: 1179–1183.
Beutler, E. 1992. Gaucher disease: new molecular approaches to diagnosis and treatment. Science 256: 794–799.
Larder, B.A. and Kemp, S.D. 1989. Multiple mutations in HIV-1 reverse transcrip-tase confer high-level resistance to zidovudine (AZT). Science 246: 1155–1158.
Omata, M., Ehata, T., Yokosuka, O., Hosoda, K., and Ohto, M. 1990. Mutations in the precore region of hepatitis B virus DNA in patients with fulminant and severe hepatitis. N. Engl. J. Med. 322: 178–183.
Tompkins, L.S. 1992. The use of molecular methods in infectious diseases. N. Engl. J. Med. 327: 1290–1297.
Lipshutz, R.J., Morris, D., Chee, M., Hubbell, E., Kozal, M.J., Shah, N. et al. 1995. Using oligonucleotide probe arrays to access genetic diversity. Biotechniques 19: 442–447.
Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K., and Sekiya, T. 1989. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Acad. Sci. USA 86: 2766–2770.
White, M.B., Carvalho, M., Derse, D., O'Brien, S.J., and Dean, M. 1992. Detecting single base substitutions as heteroduplex polymorphisms. Genomics 12: 301–306.
Nash, K.A., Gaytan, A., and Inderlied, C.B. 1997. Detection of rifampin resistance in Mycobacterium tuberculosis by use of a rapid, simple, and specific RNA/RNA mismatch assay. J. Infect. Dis. 176: 533–536.
Tyagi, S. and Kramer, F.R. 1996. Molecular beacons: probes that fluoresce upon hybridization. Bio/technology 14: 303–308.
Tyagi, S., Bratu, D.P.,and Kramer, F.R. 1998. Multicolor molecular beacons for allele discrimination. Bio/technology 16: 49–53.
Centers for Disease Control.1993. Estimates of future global tuberculosis morbidity and mortality. Morb. Mortal. Wkly. Rep. 42: 961–964.
Fischl, M.A., Uttamchandani, R.B., Daikos, G.L., Poblete, R.B., Moreno, J.N., Reyes, R.R. et al. 1992. An outbreak of tuberculosis caused by multi-drug-resistant tubercle bacilli among patients with HIV infection. Ann. Intern. Med. 117: 177–183.
Goble, M., Iseman, M.D., Madsen, L.A., Waite, D., Ackerson, L, and Horsburgh, E.R. Jr. 1993. Treatment of 171 patients with pulmonary tuberculosis resistant to isoniazid and rifampin. N. Engl. J. Med 328: 527–532.
Heifets, L.B., 1986. Rapid automated methods (BACTEC system) in clinical mycobacteriology. Semin. Respir. Infect. 1: 242–249.
Pablos-Mendez, A., Sterling, T.R., and Frieden, T.R. 1996. The relationship between delayed or incomplete treatment and all-cause mortality in patients with tuberculosis. JAMA 276: 1223–1228.
Turett, G.S., Telzak, E.E., Torian, L.V., Blum, S., Alland, D., Weisfuse, I. et al. 1995. Improved outcomes for patients with multidrug-resistant tuberculosis. Clin. Infect. Dis. 21: 1238–1244.
Telenti, A., Imboden, P., Marchesi, F. Lowrie, D., Cole, S., Colston, M.J. et al. 1993. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet 341: 647–650.
Mitchison, D. and Nunn, A. 1998. Influence of initial drug resistance on the response to short-course chemotherapy of pulmonary tuberculosis. American Review of Respiratory Diseases 133: 423–430.
Musser, J.M. 1995. Antimicrobial agent resistance in mycobacteria: molecular genetic insights. Clin. Microbiol. Rev. 8: 496–514.
Williams, D.L., Waguespack, C., Eisenach, K., Crawford, J.T., Portaels, F. Salfinger, M. et al. 1994. Characterization of rifampin resistance in pathogenic mycobacteria. Antimicrob. Agents Chemother. 38: 2380–2386.
De Beenhouwer, H., Lhiang, Z., Hannes, G. et al. 1994. Rapid detection of rifampin resistance in sputum and biopsy specimens from tuberculosis patients by PCR and line probe assay. Tuber. Lung Dis. 76: 425–430.
van Soolingen, D., Hermans, P.W.M., de Haas, P.E.W. Soil, E.R., and van Embden, J.D.A. 1991. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J. Clin. Microbiol. 29: 2578–2586.
Alland, D., Kalkut, G.E., Moss, A.R., McAdam, R.A., Hahn, J.A. Bosworth, W. et al. 1994. Transmission of tuberculosis in New York City, an analysis by DNA fingerprinting and Conventional Epidemologic Methods. N. Engl. J. Med. 330: 1710–1716.
Telenti, A., Honore, N., Bernasconi, C. March, J., Ortega, A. Heym, B. et al. 1997. Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at reference laboratory level. J. Clin. Micro. 35: 719–723.
Kent, P.R. and Kubica, G.R. 1985. Public health mycobacteriology: A guide for the level III laboratory. Atlanta: Department of Health and Human Services.
Chaulet, P., Boulahbal, F., and Grosset, J. 1995. Surveillance of drug resistance for tuberculosis control: Why and how?. Tuber. Lung Dis. 76: 487–492.
Kox, L.F.F., Van Leeuwen, J., Knijper, S. Jansen, H.M., and Kolk, A.H.J. 1995. PCR assay based on DNA coding for 16S rRNA for detection and identification of Mycobacteria in clinical samples. J. Clin. Microbiol. 35: 3225–3233.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Piatek, A., Tyagi, S., Pol, A. et al. Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nat Biotechnol 16, 359–363 (1998). https://doi.org/10.1038/nbt0498-359
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nbt0498-359
This article is cited by
-
Epidemiology of molecular probes in Xpert MTB/RIF assay in Khyber Pakhtunkhwa, Pakistan
Archives of Microbiology (2021)
-
Nucleic Acids Analysis
Science China Chemistry (2021)
-
Progress in exosome associated tumor markers and their detection methods
Molecular Biomedicine (2020)
-
A peptide nucleic acid–regulated fluorescence resonance energy transfer DNA assay based on the use of carbon dots and gold nanoparticles
Microchimica Acta (2020)
-
Next-generation fluorescent nucleic acids probes for microscopic analysis of intracellular nucleic acids
Applied Microscopy (2019)