Skip to main content
SpringerLink
Log in

Viable Real-Time PCR in Environmental Samples: Can All Data Be Interpreted Directly?

  • Notes and Short communications
  • Published:
Microbial Ecology Aims and scope Submit manuscript

Abstract

Selective nucleic acid intercalating dyes—ethidium monoazide (EMA) and propidium monoazide (PMA)—represent one of the most successful recent approaches to detect viable cells (as defined by an intact cell membrane) by PCR and have been effectively evaluated in different microorganisms. However, some practical limitations were found, especially in environmental samples. The aim of this work was to show that in the application of viable real-time PCR, there may be significant biases and to propose a strategy for overcoming some of these problems. We present an approach based on the combination of three real-time PCR amplifications for each sample that should provide an improved estimation of the number of viable cells. This approach could be useful especially when it is difficult to determine a priori how to optimize methods using PMA or EMA. Although further studies are required to improve viable real-time PCR methods, the concept as outlined here presents an interesting future research direction.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2

References

  1. AFNOR XP T90-471 (2006) Détection et quantification des Legionella et/ou Legionella pneumophila par concentration et amplification génique par reaction de polymérisation en chaîne (PCR). Normalization Française

  2. Bae S, Wuertz S (2009) Discrimination of viable and dead fecal bacteroidales bacteria by quantitative PCR with propidium monoazide. Appl Environ Microbiol 75:2940–2944

    Article  CAS  PubMed  Google Scholar 

  3. Behets J, Declerck P, Delaedt Y, Creemers B, Ollevier F (2007) Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples. J Microbiol Methods 68:137–144

    Article  CAS  PubMed  Google Scholar 

  4. Bolton PH, Kearns DR (1978) Spectroscopic properties of ethidium monoazide: a fluorescent photoaffinity label for nucleic acids. Nucleic Acids Res 5:4981–4903

    Article  Google Scholar 

  5. Cawthorn DM, Witthuhn RC (2008) Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide. J Appl Microbiol 105:1178–1185

    Article  CAS  PubMed  Google Scholar 

  6. Chang B, Taguri T, Sugiyama K, Amemura-Maekawa J, Kura F, Watanabe H (2010) Comparison of ethidium monoazide and propidium monoazide for the selective detection of viable Legionella cells. Jpn J Infect Dis 63:119–123

    CAS  PubMed  Google Scholar 

  7. Delgado Viscogliosi P, Solignac L, Delattre JM (2009) Viability PCR, a culture-independent method for rapid and selective quantification of viable Legionella pneumophila cells in environmental water samples. Appl Environ Microbiol 75:3502–3512

    Article  CAS  PubMed  Google Scholar 

  8. Flekna G, Stefanic P, Wagner M, Smulders FJ, Mozina SS, Hein I (2007) Insufficient differentiation of live and dead Campylobacter jejuni and Listeria monocytogenes cells by ethidium monoazide (EMA) compromises EMA/real-time PCR. Res Microbiol 158:405–412

    Article  CAS  PubMed  Google Scholar 

  9. Graiver DA, Saunders SE, Topliff CL, Kelling CL, Bartelt-Hunt SL (2010) Ethidium monoazide does not inhibit RT-PCR amplification of nonviable avian influenza RNA. J Virol Methods 164:51–54. doi:10.1016/j.jviromet.2009.11.024

    Article  CAS  PubMed  Google Scholar 

  10. Kobayashi H, Oethinger M, Tuohy MJ, Hall GS, Bauer TW (2009) Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermis by ethidium bromide monoazide. Lett Appl Microbiol 48:633–638

    Article  CAS  PubMed  Google Scholar 

  11. Kralik P, Nocker A, Pavlik I (2010) Mycobacterium avium subsp. paratuberculosis viability determination using F57 quantitative PCR in combination with propidium monoazide treatment. Int J Food Microbiol. doi:10.1016/j.ijfoodmicro.2010.03.018

  12. Nocker A, Camper AK (2006) Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. Appl Environ Microbiol 72:1997–2004

    Article  CAS  PubMed  Google Scholar 

  13. Nocker A, Camper AK (2009) Novel approaches toward preferential detection of viable cells using nucleic acid amplification techniques. FEMS Microbiol Lett 291:137–142

    Article  CAS  PubMed  Google Scholar 

  14. Nocker A, Cheung CY, Camper AK (2006) Comparison of propidium monoazide and ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J Microbiol Meth 67:310–320

    Article  CAS  Google Scholar 

  15. Nocker A, Sossa-Fernandez P, Burr MD, Camper AK (2007) Use of propidium monoazide for live/dead distinction in microbial ecology. Appl Environ Microbiol 73:5111–5117

    Article  CAS  PubMed  Google Scholar 

  16. Nocker A, Mazza A, Masson L, Camper AK, Brousseau R (2009) Selective detection of live bacteria combining propidium monoazide sample treatment with microarray technology. J Microbiol Methods 76:253–261

    Article  CAS  PubMed  Google Scholar 

  17. Nogva HK, Dromtorp SM, Nissen H, Rudi K (2003) Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5′-nuclease PCR. Biotechniques 810:812–813

    Google Scholar 

  18. Pan Y, Breidt F (2007) Enumeration of viable Listeria monocytogenes cells by real-time PCR with propidium monoazide and ethidium monoazide in the presence of dead cells. Appl Environ Microbiol 73:8028–8031

    Article  CAS  PubMed  Google Scholar 

  19. Pisz JM, Lawrence JR, Schafer AN, Siciliano SD (2007) Differentiation of genes extracted from nonviable versus viable micro-organisms in environmental samples using ethidium monoazide bromide. J Microbiol Methods 71:312–318

    Article  CAS  PubMed  Google Scholar 

  20. Rasmussen R (2001) Quantification on the LightCycler. In: Meuer S, Wittwer C, Nakagawara K (eds) Rapid cycle real-time PCR, methods and applications. Springer, Heidelberg, pp 21–34

    Google Scholar 

  21. Rawsthorne H, Dock CN, Jaykus LA (2009) PCR-based method using propidium monoazide to distinguish viable from nonviable Bacillus subtilis spores. Appl Environ Microbiol 75:2936–2939

    Article  CAS  PubMed  Google Scholar 

  22. Rudi K, Moen B, Drømtorp SM, Holck L (2005) Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples. Appl Environ Microbiol 71:1018–1024

    Article  CAS  PubMed  Google Scholar 

  23. Soejima T, Iida K, Qin T, Taniai H, Seki M, Takade A, Yoshida S (2007) Photoactivated ethidium monoazide directly cleaves bacterial DNA and is applied to PCR for discrimination of live and dead bacteria. Microbiol Immunol 51:763–775

    CAS  PubMed  Google Scholar 

  24. Varma M, Field R, Stinson M, Rukovets B, Wymer L, Haugland R (2009) Quantitative real-time PCR analysis of total and propidium monoazide-resistant fecal indicator bacteria in wastewater. Water Res 43:4790–4801. doi:10.1016/j.watres.2009.05.03

    Article  CAS  PubMed  Google Scholar 

  25. Vesper S, McKinstry C, Hartmann C, Neace M, Yoder S, Vesper A (2008) Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA). J Microbiol Methods 72:180–184

    Article  CAS  PubMed  Google Scholar 

  26. Wagner AO, Malin C, Knapp BA, Illmer P (2008) Removal of free extracellular DNA from environmental samples by ethidium monoazide and propidium monoazide. Appl Environ Microbiol 74:2537–2539

    Article  CAS  PubMed  Google Scholar 

  27. Wang S, Levin RE (2006) Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR. J Microbiol Methods 64:1–8

    Article  CAS  PubMed  Google Scholar 

  28. Wang L, Li Y, Mustapha A (2009) Detection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCR. J Appl Microbiol 107:1719–1728

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We are most grateful to the Comissionat per a Universitats i Recerca del Departament d’Innovació, Universitats i Empresa de la Generalitat de Catalunya i del Fons Social Europeu for the financial support received from the program of pre-doctoral scholarships FI and the program of Beques per a estades de recerca fora de Catalunya (BE 2008). This work was supported by Agència Catalana de Seguretat Alimentària and by the Ministerio de Ciencia e Innovación with grant CTM2008-06676-C05-02/TECNO to Jordi Morató.

Author information

Authors and Affiliations

  1. Laboratori de Microbiologia Sanitària i Mediambiental (MSMLab)-Aquasost, UNESCO Chair in Sustainability, Universitat Politècnica de Catalunya, C/Violinista Vellsolà 37, 08222, Terrassa, Barcelona, Spain

    Mariana Fittipaldi, Francesc Codony, Barbara Adrados & Jordi Morató

  2. Center for Biofilm Engineering, Montana State University, 366 EPS Building, Bozeman, MT, USA

    Mariana Fittipaldi & Anne K Camper

Authors
  1. Mariana Fittipaldi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Francesc Codony
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Barbara Adrados
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anne K Camper
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jordi Morató
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Francesc Codony.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fittipaldi, M., Codony, F., Adrados, B. et al. Viable Real-Time PCR in Environmental Samples: Can All Data Be Interpreted Directly?. Microb Ecol 61, 7–12 (2011). https://doi.org/10.1007/s00248-010-9719-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00248-010-9719-1

Keywords

Search

Navigation