Simultaneous detection of Legionella species and Legionella pneumophila by duplex PCR (dPCR) assay in cooling tower water samples from Jakarta, Indonesia

  • Andi Yasmon
  • Yusmaniar Yusmaniar
  • Anis Anis
  • Budiman Bela
Keywords: BCYE media, mip gene, 16S-rRNA gene
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Aim: Since culture method is time-consuming and has low sensitivity, we developed a duplex PCR (dPCR) assay for the detection of Legionella sp. and L. pneumophila in cooling tower samples. We used culture method as a gold standard.

Methods: Optimization of dPCR method was performed to obtain an assay with high sensitivity and specifi city. The optimized method was used to detect Legionella sp. dan L. pneumophila in 9 samples obtained from 9 buildings in Jakarta. For culture method, the bacteria were grown or isolated on selective growth factor supplemented-buffered charcoal yeast extract (BCYE) media.

Results: Of 9 samples tested by dPCR assay, 6 were positive for Legionella species,1 was positive for L. pneumophila, and 2 showed negative results. For the same samples, no Legionella sp. was detected by the culture method.

Conclusion: dPCR assay was much more sensitive than the culture method and was potentially used as a rapid, specifi c and sensitive test for routine detection of Legionella sp. dan for L. pneumophila in water samples. (Med J Indones 2010; 19:223-7)


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  1. Pedro-Botet ML, Stout JE, Yu VL. Legionnaires' disease contracted from patient homes: the coming of the third plague? Eur J Clin Microbiol Infect Dis. 2002;21:699-705.

  2. Palusinska-Szysz M, Cendrowska-Pinkosz M. Pathogenicity of the family Legionellaceae. Arch Immunol Ther Exp (Warsz). 2009;57:279-90.

  3. Diederen BM. Legionella spp. and Legionnaires' disease. J Infect. 2008;56:1-12.

  4. Fields BS, Benson RF, Besser RE. Legionella and Legionnaires' disease: 25 years of investigation. Clin Microbiol Rev. 2002;15:506-26.

  5. Devos L, Boon N, Verstraete W. Legionella pneumophila in the environment: The occurrence of a fastidious bacterium in oligotrophic conditions. Rev Environ Sci Biotechnol. 2005;4:61-74.

  6. Gobin I, Susa M, Begic G, Hartland EL, Doric M. Experimental Legionella longbeachae infection in intratracheally inoculated mice. J Med Microbiol. 2009;58:723-30.

  7. Bollin GE, Plouffe JF, Para MF, Hackman B. Aerosols containing Legionella pneumophila generated by shower heads and hot-water faucets. Appl Environ Microbiol. 1985;50:1128-31.

  8. Hsu BM, Chen CH, Wan MT, Cheng HW. Legionella prevalence in hot spring recreation areas of Taiwan. Water Res. 2006;40:3267-73.

  9. Ragull S, Garcia-Nunez M, Pedro-Botet ML, Sopena N, Esteve M, Montenegro R, et al. Legionella pneumophila in cooling towers: fl uctuations in counts, determination of genetic variability by pulsed-fi eld gel electrophoresis (PFGE), and persistence of PFGE patterns. Appl Environ Microbiol. 2007;73:5382-4.

  10. Levi K, Smedley J, Towner KJ. Evaluation of a real-time PCR hybridization assay for rapid detection of Legionella pneumophila in hospital and environmental water samples. Clin Microbiol Infect. 2003;9:754-8.

  11. Koide M, Saito A, Kusano N, Higa F. Detection of Legionella spp. in cooling tower water by the polymerase chain reaction method. Appl Environ Microbiol. 1993;59:1943-6.

  12. Nintasen R, Utrarachkij F, Siripanichgon K, Bhumiratana A, Suzuki Y, Suthienkul O. Enhancement of Legionella pneumophila culture isolation from microenvironments by macrophage infectivity potentiator (mip) gene-specifi c nested polymerase chain reaction. Microbiol Immunol. 2007;51:777-85.

  13. Joly P, Falconnet PA, Andre J, Weill N, Reyrolle M, Vandenesch F, et al. Quantitative real-time Legionella PCR for environmental water samples: data interpretation. Appl Environ Microbiol. 2006;72:2801-8.

  14. Devos L, Clymans K, Boon N, Verstraete W. Evaluation of nested PCR assays for the detection of Legionella pneumophila in a wide range of aquatic samples. J Appl Microbiol. 2005;99:916-25.

  15. Templeton KE, Scheltinga SA, Sillekens P, Crielaard JW, van Dam AP, Goossens H, et al. Development and clinical evaluation of an internally controlled, single-tube multiplex real-time PCR assay for detection of Legionella pneumophila and other Legionella species. J Clin Microbiol. 2003;41:4016-21.

  16. England AC, Fraser DW, Mallison GF, Mackel DC, Skaliy P, Gorman GW. Failure of Legionella pneumophila sensitivities to predict culture results from disinfectant-treated air-conditioning cooling towers. Appl Environ Microbiol. 1982;43:240-4.

  17. Ohno A, Kato N, Yamada K, Yamaguchi K. Factors infl uencing survival of Legionella pneumophila serotype 1 in hot spring water and tap water. Appl Environ Microbiol. 2003;69:2540-7.

  18. Declerck P, Verelst L, Duvivier L, Van Damme A, Ollevier F. PCR as a test for the presence or absence of Legionella in (cooling) water. Water Sci Technol. 2003;47:103-7.

  19. Delgado-Viscogliosi P, Solignac L, Delattre JM. Viability PCR, a culture-independent method for rapid and selective quantifi cation of viable Legionella pneumophila cells in environmental water samples. Appl Environ Microbiol. 2009;75:3502-12.

How to Cite
Yasmon A, Yusmaniar Y, Anis A, Bela B. Simultaneous detection of Legionella species and Legionella pneumophila by duplex PCR (dPCR) assay in cooling tower water samples from Jakarta, Indonesia. Med J Indones [Internet]. 2010Nov.1 [cited 2024Jun.18];19(4):223-7. Available from:
Basic Medical Research

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