Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Free

Abstract

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of was developed and evaluated. The assay specifically amplified only sequences, and no cross-reactivity was observed for other species or respiratory bacterial species. The detection limit for this assay was found to be 2 × 10 copies, corresponding to 2–20 colour changing units of in 1 h, as observed in a real-time turbidimeter and electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis as well as direct sequencing of the amplified product. The assay was applied to 95 nasopharyngeal swab samples collected from patients or from healthy individuals, and compared to a real-time PCR assay in-house. A concordance of 100 % was observed between the two assays. The LAMP assay is easy to perform, shows a rapid reaction and is inexpensive. It may therefore be applied in the routine diagnosis of infection in the clinical laboratory.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.46071-0
2005-11-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jmm/54/11/JM541106.html?itemId=/content/journal/jmm/10.1099/jmm.0.46071-0&mimeType=html&fmt=ahah

References

  1. Abele-Horn M., Busch U., Nitschko H., Jacobs E., Bax R., Pfaff F., Schaffer B., Heesemann J. 1998; Molecular approaches to diagnosis of pulmonary diseases due to Mycoplasma pneumoniae . J Clin Microbiol 36:548–551
    [Google Scholar]
  2. Chia W. K., Spence L., Dunkley L., Bradbury W. 1988; Development of urease conjugated enzyme-linked immunosorbent assays (ELISA) for the detection of IgM and IgG antibodies against Mycoplasma pneumoniae in human sera. Diagn Microbiol Infect Dis 11:101–107 [CrossRef]
    [Google Scholar]
  3. Clyde W. A. Jr 1993; Clinical overview of typical Mycoplasma pneumoniae infections. Clin Infect Dis 17 (Suppl. 1)S32–S37
    [Google Scholar]
  4. Dorigo-Zetsma J. W., Zaat S. A., Wertheim-van Dillen P. M., Spanjaard L., Rijntjes J., Van Waveren G., Jensen J. S., Angulo A. F., Dankert J. 1999; Comparison of PCR, culture, and serological tests for diagnosis of Mycoplasma pneumoniae respiratory tract infection in children. J Clin Microbiol 37:14–17
    [Google Scholar]
  5. Fedorko D. P., Emery D. D., Franklin S. M., Congdon D. D. 1995; Evaluation of a rapid enzyme immunoassay system for serologic diagnosis of Mycoplasma pneumoniae infection. Diagn Microbiol Infect Dis 23:85–88 [CrossRef]
    [Google Scholar]
  6. Foy H. M. 1993; Infections caused by Mycoplasma pneumoniae and possible carrier state in different populations of patients. Clin Infect Dis 17 (Suppl. 1):S37–S47
    [Google Scholar]
  7. Hardegger D., Nadal D., Bossart W., Altwegg M., Dutly F. 2000; Rapid detection of Mycoplasma pneumoniae in clinical samples by real-time PCR. J Microbiol Methods 41:45–51 [CrossRef]
    [Google Scholar]
  8. Harris R., Marmion B. P., Varkanis G., Kok T., Lunn B., Martin J. 1988; Laboratory diagnosis of Mycoplasma pneumoniae infection.2. Comparison of methods for the direct detection of specific antigen or nucleic acid sequences in respiratory exudates. Epidemiol Infect 101:685–694 [CrossRef]
    [Google Scholar]
  9. Hong T. C., Mai Q. L., Cuong D. V., Parida M., Minekawa H., Notomi T., Hasebe F., Morita K. 2004; Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus. J Clin Microbiol 42:1956–1961 [CrossRef]
    [Google Scholar]
  10. Ieven M., Ursi D., Van Bever H., Quint W., Niesters H. G., Goossens H. 1996; Detection of Mycoplasma pneumoniae by two polymerase chain reactions and role of M.pneumoniae in acute respiratory tract infections in pediatric patients. J Infect Dis 173:1445–1452 [CrossRef]
    [Google Scholar]
  11. Iwamoto T., Sonobe T., Hayashi K. 2003; Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M.avium , and M. intracellulare in sputum samples. J Clin Microbiol 41:2616–2622 [CrossRef]
    [Google Scholar]
  12. Kok T. W., Varkanis G., Marmion B. P., Martin J., Esterman A. 1988; Laboratory diagnosis of Mycoplasma pneumoniae infection.1. Direct detection of antigen in respiratory exudates by enzyme immunoassay. Epidemiol Infect 101:669–684 [CrossRef]
    [Google Scholar]
  13. Loens K., Ursi D., Ieven M., Van Aarle P., Sillekens P., Oudshoorn P., Goossens H. 2002; Detection of Mycoplasma pneumoniae in spiked clinical samples by nucleic acid sequence-based amplification. J Clin Microbiol 40:1339–1345 [CrossRef]
    [Google Scholar]
  14. Mori Y., Nagamine K., Tomita N., Notomi T. 2001; Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 289:150–154 [CrossRef]
    [Google Scholar]
  15. Notomi T., Okayama H., Masubuchi H., Yonekawa T., Watanabe K., Amino N., Hase T. 2000; Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63– E63 [CrossRef]
    [Google Scholar]
  16. Sillis M. 1990; The limitations of IgM assays in the serological diagnosis of Mycoplasma pneumoniae infections. J Med Microbiol 33:253–258 [CrossRef]
    [Google Scholar]
  17. Templeton K. E., Scheltinga S. A., Graffelman A. W. & 7 other authors; 2003; Comparison and evaluation of real-time PCR, real-time nucleic acid sequence-based amplification, conventional PCR, and serology for diagnosis of Mycoplasma pneumoniae . J Clin Microbiol 41:4366–4371 [CrossRef]
    [Google Scholar]
  18. Tjhie J. H., Van Kuppeveld F. J., Roosendaal R., Melchers W. J., Gordijn R., MacLaren D. M., Walboomers J. M., Meijer C. J., van den Brule A. J. 1994; Direct PCR enables detection of Mycoplasma pneumoniae in patients with respiratory tract infections. J Clin Microbiol 32:11–16
    [Google Scholar]
  19. Ursi D., Dirven K., Loens K., Ieven M., Goossens H. 2003; Detection of Mycoplasma pneumoniae in respiratory samples by real-time PCR using an inhibition control. J Microbiol Methods 55:149–153 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.46071-0
Loading
/content/journal/jmm/10.1099/jmm.0.46071-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed