1887

Abstract

Loop-mediated isothermal amplification (LAMP) is a novel, rapid nucleic acid amplification method with high specificity and sensitivity under isothermal conditions. In this study a LAMP assay for diagnosing pneumonia (PCP) was developed. Oligonucleotide primers specific for species were designed corresponding to 18S rRNA gene sequences. The assay, performed for 30 min at 61 °C, was capable of detecting 50 copies per tube (2×10 copies ml) in 30 min and did not show cross-reactivity to other species of fungi, including the genera , and . A total of 21 of 24 clinical specimens (sputum and bronchoalveolar lavage fluid) from patients with suspected PCP tested positive using the LAMP assay by real-time fluorescence detection. The results of the LAMP reaction were also observed by real-time turbidity detection and end-point visual turbidity or fluorescence detection. With real-time fluorescence detection, melting curves of the products were effective at distinguishing specific amplification from non-specific amplification or self-amplification. Visual detection was also possible as a rapid and easy assay using only a heat block and a black light.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.47216-0
2008-01-01
2019-10-18
Loading full text...

Full text loading...

/deliver/fulltext/jmm/57/1/50.html?itemId=/content/journal/jmm/10.1099/jmm.0.47216-0&mimeType=html&fmt=ahah

References

  1. Brancart, F., Rodriguez-Villalobos, H., Fonteyne, P. A., Peres-Bota, D. & Liesnard, C. ( 2005; ). Quantitative TaqMan PCR for detection of Pneumocystis jiroveci. J Microbiol Methods 61, 381–387.[CrossRef]
    [Google Scholar]
  2. Bu, R., Sathiapalan, R. K., Ibrahim, M. M., Al-Mohsen, I., Almodavar, E., Gutierrez, M. I. & Bhatia, K. ( 2005; ). Monochrome LightCycler PCR assay for detection and quantification of five common species of Candida and Aspergillus. J Med Microbiol 54, 243–248.[CrossRef]
    [Google Scholar]
  3. Cenis, J. L. ( 1992; ). Rapid extraction of fungal DNA for PCR amplification. Nucleic Acids Res 20, 2380 [CrossRef]
    [Google Scholar]
  4. Endo, S., Komori, T., Ricci, G., Sano, A., Yokoyama, K., Ohori, A., Kamei, K., Franco, M., Miyaji, M. & Nishimura, K. ( 2004; ). Detection of gp43 of Paracoccidioides brasiliensis by the loop-mediated isothermal amplification (LAMP) method. FEMS Microbiol Lett 234, 93–97.[CrossRef]
    [Google Scholar]
  5. Fischer, S., Gill, V. J., Kovacs, J., Miele, P., Keary, J., Silcott, V., Huang, S., Borio, L., Stock, F. & other authors ( 2001; ). The use of oral washes to diagnose Pneumocystis carinii pneumonia: a blinded prospective study using a polymerase chain reaction-based detection system. J Infect Dis 184, 1485–1488.[CrossRef]
    [Google Scholar]
  6. Flori, P., Bellete, B., Durand, F., Raberin, H., Cazorla, C., Hafid, J., Lucht, F. & Sung, R. T. ( 2004; ). Comparison between real-time PCR, conventional PCR and different staining techniques for diagnosing Pneumocystis jiroveci pneumonia from bronchoalveolar lavage specimens. J Med Microbiol 53, 603–607.[CrossRef]
    [Google Scholar]
  7. Frenkel, J. K. ( 1999; ). Pneumocystis pneumonia, an immunodeficiency-dependent disease (IDD): a critical historical overview. J Eukaryot Microbiol 46, 89S–92S.
    [Google Scholar]
  8. Haverstick, D. M., Bullock, G. C. & Bruns, D. E. ( 2004; ). Genotyping of hepatitis C virus by melting curve analysis: analytical characteristics and performance. Clin Chem 50, 2405–2407.[CrossRef]
    [Google Scholar]
  9. Helweg-Larsen, J., Tsolaki, A. G., Miller, R. F., Lundgren, B. & Wakefield, A. E. ( 1998; ). Clusters of Pneumocystis carinii pneumonia: analysis of person-to-person transmission by genotyping. QJM 91, 813–820.[CrossRef]
    [Google Scholar]
  10. Hocker, B., Wendt, C., Nahimana, A., Tonshoff, B. & Hauser, P. M. ( 2005; ). Molecular evidence of Pneumocystis transmission in pediatric transplant unit. Emerg Infect Dis 11, 330–332.[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. Jaijakul, S., Saksirisampant, W., Prownebon, J., Yenthakam, S., Mungthin, M., Leelayoova, S. & Nuchprayoon, S. ( 2005; ). Pneumocystis jiroveci in HIV/AIDS patients: detection by FTA filter paper together with PCR in noninvasive induced sputum specimens. J Med Assoc Thai 88, S294–S299.
    [Google Scholar]
  13. Larsen, H. H., Masur, H., Kovacs, J. A., Gill, V. J., Silcott, V. A., Kogulan, P., Maenza, J., Smith, M., Lucey, D. R. & Fischer, S. H. ( 2002; ). Development and evaluation of a quantitative, touch-down, real-time PCR assay for diagnosing Pneumocystis carinii pneumonia. J Clin Microbiol 40, 490–494.[CrossRef]
    [Google Scholar]
  14. Makimura, K., Murayama, S. Y. & Yamaguchi, H. ( 1994; ). Detection of a wide range of medically important fungi by the polymerase chain reaction. J Med Microbiol 40, 358–364.[CrossRef]
    [Google Scholar]
  15. Maskell, N. A., Waine, D. J., Lindley, A., Pepperell, J. C., Wakefield, A. E., Miller, R. F. & Davies, R. J. ( 2003; ). Asymptomatic carriage of Pneumocystis jiroveci in subjects undergoing bronchoscopy: a prospective study. Thorax 58, 594–597.[CrossRef]
    [Google Scholar]
  16. Medrano, F. J., Montes-Cano, M., Conde, M., de la Horra, C., Respaldiza, N., Gasch, A., Perez-Lozano, M. J., Varela, J. M. & Calderon, E. J. ( 2005; ). Pneumocystis jirovecii in general population. Emerg Infect Dis 11, 245–250.[CrossRef]
    [Google Scholar]
  17. 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]
  18. Nagamine, K., Hase, T. & Notomi, T. ( 2002; ). Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16, 223–229.[CrossRef]
    [Google Scholar]
  19. 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 [CrossRef]
    [Google Scholar]
  20. Poon, L. L., Leung, C. S., Chan, K. H., Lee, J. H., Yuen, K. Y., Guan, Y. & Peiris, J. S. ( 2005; ). Detection of human influenza A viruses by loop-mediated isothermal amplification. J Clin Microbiol 43, 427–430.[CrossRef]
    [Google Scholar]
  21. Poon, L. L., Wong, B. W., Ma, E. H., Chan, K. H., Chow, L. M., Abeyewickreme, W., Tangpukdee, N., Yuen, K. Y., Guan, Y. & other authors ( 2006; ). Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin Chem 52, 303–306.
    [Google Scholar]
  22. Shah, J. S., Pieciak, W., Liu, J., Buharin, A. & Lane, D. J. ( 1996; ). Diversity of host species and strains of Pneumocystis carinii is based on rRNA sequences. Clin Diagn Lab Immunol 3, 119–127.
    [Google Scholar]
  23. Shrestha, N. K., Tuohy, M. J., Hall, G. S., Reischl, U., Gordon, S. M. & Procop, G. W. ( 2003; ). Detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacterial isolates by real-time PCR. J Clin Microbiol 41, 5121–5126.[CrossRef]
    [Google Scholar]
  24. Stringer, J. R., Beard, C. B., Miller, R. F. & Wakefield, A. E. ( 2002; ). A new name (Pneumocystis jirovecii) for pneumocystis from humans. Emerg Infect Dis 8, 891–896.[CrossRef]
    [Google Scholar]
  25. Vidal, S., de la Horra, C., Martin, J., Montes-Cano, M. A., Rodriguez, E., Respaldiza, N., Rodriguez, F., Varela, J. M., Medrano, F. J. & other authors ( 2006; ). Pneumocystis jirovecii colonisation in patients with interstitial lung disease. Clin Microbiol Infect 12, 231–235.[CrossRef]
    [Google Scholar]
  26. White, T. J., Bruns, T., Lee, S. & Taylor, J. ( 1990; ). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: a Guide to Methods and Applications, pp. 315–322. Edited by M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White. San Diego, CA: Academic Press.
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.47216-0
Loading
/content/journal/jmm/10.1099/jmm.0.47216-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error