Skip to main content
SpringerLink
Log in

Rapid Detection of the Escherichia coli Genospecies in Water by Conventional and Real-Time PCR

  • Protocol
  • First Online:
PCR Detection of Microbial Pathogens

Part of the book series: Methods in Molecular Biology ((MIMB,volume 943))

Abstract

The presence of Escherichia coli has long been established as the most reliable microbiological indication of fecal contamination in water. Current recommended culture-based methods for assessing water quality by the detection of E. coli are lengthy and lack ubiquity (ability to detect most if not all strains of a target microorganism). We describe rapid and sensitive conventional and real-time PCR assays specific to E. coli and Shigella, based on the nucleotide sequence of the highly conserved elongation factor Tu (tuf) gene enabling the detection of all members of the genospecies.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. United States Environmental Protection Agency. (1993) Preventing waterborne disease - A focus on EPA’s research, EPA/640/K-93/001, Office of Research and Development, United States Environmental Protection Agency, Washington, DC, 20 pages.

    Google Scholar 

  2. Sinton LW, Finlay RK, Hannah DJ (1998) Distinguishing human from faecal contamination in water: a review. N Z J Mar Freshwater Res 32:323–348

    Article  Google Scholar 

  3. Edberg SC, Rice EW, Karlin RJ, Allen MJ (2000) Escherichia coli: the best biological water indicator for public health protection. J Appl Microbiol 88 suppl:S106–S116

    Google Scholar 

  4. Doyle MP, Erickson MC (2006) Closing the door on the fecal coliform assay. Microbe 1:162–163

    Google Scholar 

  5. United States Environmental Protection Agency. (1986) Ambient water quality criteria for bacteria - 1986, EPA440/5-84-002, Office of Water Regulations and Standards Criteria and Standards Division, United States Environmental Protection Agency, Washington DC, 18 pages.

    Google Scholar 

  6. Rompré A, Servais P, Baudart J, de-Roubin M-R, Laurent P (2002) Detection and enumeration of coliforms in drinking water: current methods and emerging approaches. J Microbiol Meth 49:31–54

    Article  Google Scholar 

  7. Doyle MP, Schoeni JL (1984) Survival and growth characteristics of Escherichia coli associated with hemorrhagic colitis. Appl Environ Microbiol 48:855–856

    PubMed  CAS  Google Scholar 

  8. Chang GW, Brill J, Lum R (1989) Proportion of β-D-glucuronidase-negative Escherichia coli in human fecal samples. Appl Environ Microbiol 55:335–339

    PubMed  CAS  Google Scholar 

  9. Feng P, Lum R, Chang GW (1991) Identification of uidA gene sequences in β-D-glucuronidase-negative Escherichia coli. Appl Environ Microbiol 57:320–323

    PubMed  CAS  Google Scholar 

  10. Maheux AF, Huppé V, Boissinot M, Picard FJ, Bissonnette L, Bernier J-LT, Bergeron MG (2008) Analytical limits of four β-glucuronidase and β-galactosidase-based commercial culture methods used to detect Escherichia coli and total coliforms. J Microbiol Meth 75:506–514

    Article  CAS  Google Scholar 

  11. Boissinot M, Bergeron MG (2002) Toward rapid real-time molecular diagnostic to guide smart use of antimicrobials. Curr Opin Microbiol 5:478–482

    Article  PubMed  Google Scholar 

  12. Picard FJ, Bergeron MG (2002) Rapid molecular theranostics in infectious diseases, Drug Discov. Today 7:1092–1100

    CAS  Google Scholar 

  13. Gilbride KA, Lee D-Y, Beaudette LA (2006) Molecular techniques in wastewater: understanding microbial communities, and real-time process control. J Microbiol Meth 66:1–20

    Article  CAS  Google Scholar 

  14. Santo Domingo JW, Bambic DG, Edge TA, Wuertz S (2007) Quo vadis source tracking? Towards a strategic framework for environmental monitoring of fecal pollution. Water Res 41:3539–3552

    Article  PubMed  CAS  Google Scholar 

  15. Lawrence JG, Ochman H, Hartl DL (1991) Molecular and evolutionary relationships among enteric bacteria. J Gen Microbiol 137:1911–1921

    Article  PubMed  CAS  Google Scholar 

  16. Nataro JP, Kaper JB (1998) Diarrheagenic Escherichia coli. Clin Microbiol Rev 11:142–201

    PubMed  CAS  Google Scholar 

  17. Pupo GM, Lan R, Reeves PR (2000) Multiple independent origins of Shigella clones of Escherichia coli and convergent evolution of many of their characteristics. Proc Natl Acad Sci USA 97:10567–10572

    Article  PubMed  CAS  Google Scholar 

  18. Fukushima M, Kakinuma K, Kawaguchi R (2002) Phylogenetic analysis of Salmonella, Shigella, and Escherichia coli strains on the basis of gyrB gene sequence. J Clin Microbiol 40:2779–2785

    Article  PubMed  CAS  Google Scholar 

  19. Lan R, Reeves PR (2002) Escherichia coli in disguise: molecular origins of Shigella. Microbes Infect 4:1125–1132

    Article  PubMed  CAS  Google Scholar 

  20. Paradis S, Boissinot M, Paquette N, Bélanger SD, Martel EA, Boudreau DK, Picard FJ, Ouellette M, Roy PH, Bergeron MG (2005) Phylogeny of the Enterobacteriaceae based on genes encoding elongation factor Tu and F-ATPase β-subunit. Int J Syst Evol Microbiol 55:2013–2025

    Article  PubMed  CAS  Google Scholar 

  21. Touchon M, Hoede C, Tenaillon O, Barbe V, Baeriswyl S, Bidet P, Bingen E, Bonacorsi S, Bouchier C, Bouvet O, Calteau A, Chiapello H, Clermont O, Cruveiller S, Danchin A, Diard M, Dossat C, Karoui ME, Frapy E, Garry L, Ghigo JM, Gilles AM, Johnson J, Le Bouguénec C, Lescat M, Mangenot S, Martinez-Jéhanne V, Matic I, Nassif X, Oztas S, Petit MA, Pichon C, Rouy Z, Ruf CS, Schneider D, Tourret J, Vacherie B, Vallenet D, Médigue C, Rocha EP, Denamur E (2009) Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths. PLoS Genet 5:e1000344

    Article  PubMed  Google Scholar 

  22. Brenner DJ, Fanning GR, Skerman FJ, Falkow S (1972) Polynucleotide sequence divergence among strains of Escherichia coli and closely related organisms. J Bacteriol 109:953–965

    PubMed  CAS  Google Scholar 

  23. Brenner DJ, Fanning GR, Miklos GV, Steigerwalt AG (1973) Polynucleotide sequence relatedness among Shigella species. Int J Syst Bacteriol 23:1–7

    Article  Google Scholar 

  24. Brenner DG (1984) Family I Enterobacteriaceae Rhan (1937). In: Krieg NR, Holt JG (eds) Bergey’s Manual of systematic bacteriology, vol 1. The Williams & Williams Co, Baltimore, pp 410–411

    Google Scholar 

  25. Maheux AF, Picard FJ, Boissinot M, Bissonnette L, Paradis S, Bergeron MG (2009) Analytical comparison of nine PCR primer sets designed to detect the presence of Escherichia coli/Shigella in water samples. Water Res 43:3019–3028

    Article  PubMed  CAS  Google Scholar 

  26. Picard FJ, Gagnon M, Bernier MR, Parham NJ, Bastien M, Boissinot M, Peytavi R, Bergeron MG (2009) Internal control for nucleic acid testing based on the use of purified Bacillus atrophaeus subsp. globigii spores. J Clin Microbiol 47:751–757

    Article  PubMed  CAS  Google Scholar 

  27. Jordan JA (2000) Real-time detection of PCR products and microbiology. New Technol Life Sci 6:61–66

    Google Scholar 

  28. Yang S, Rothman RE (2004) PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect Dis 4:337–348

    Article  PubMed  CAS  Google Scholar 

  29. Picard FJ, Ménard C, Bastien M, Boissinot M (2005-02-17) Method for the preparation of reagents for amplification and/or detection of nucleic acids that exhibit no significant contamination by nucleic acids, United States Patent Application 20050037349.

    Google Scholar 

  30. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  PubMed  CAS  Google Scholar 

  31. United States Environmental Protection Agency. (2004) Quality assurance/quality control guidance for laboratories performing PCR analyses on environmental samples, EPA 815-B-04-001. Office of Water (4607), United States Environmental Protection Agency, Cincinnati, OH, 56 pages.

    Google Scholar 

  32. Mitchell PS, Germer JJ, Patel R (2004) Nucleic acid amplification methods: laboratory design and operations. In: Persing DH, Tenover FC, Versalovic J, Tang Y-W, Unger ER, Relman DA, White TJ (eds) Molecular microbiology: Diagnostic principles and practice. ASM Press, Washington, DC, pp 85–93

    Google Scholar 

  33. Glushkov SA, Bragin AG, Dymshits GM (2009) Decontamination of polymerase chain reaction reagents using DEAE-cellulose. Anal Biochem 393:135–137

    Article  PubMed  CAS  Google Scholar 

  34. Shepley DP, Wolk DM (2004) Quantitative molecular methods: result standardization, interpretation, and laboratory quality control. In: Persing DH, Tenover FC, Versalovic J, Tang Y-W, Unger ER, Relman DA, White TJ (eds) Molecular microbiology: Diagnostic principles and practice. ASM Press, Washington, DC, pp 95–129

    Google Scholar 

Download references

Acknowledgments

We thank Dr Maurice Boissinot for critical reading of the manuscript.

Author information

Authors and Affiliations

  1. Département de Microbiologie-infectiologie et Immunologie, Faculté de médecine, Centre de Recherche en Infectiologie, Centre de Recherche du CHUQ, Université Laval, Québec, Canada

    Andrée F. Maheux & Michel G. Bergeron

  2. Département de Microbiologie-infectiologie et Immunologie, Faculté de Médecine, Centre de Recherche en Infectiologie, Centre de Recherche du CHUQ, Université Laval, Québec, Canada

    Luc Bissonnette

Authors
  1. Andrée F. Maheux
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Luc Bissonnette
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michel G. Bergeron
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michel G. Bergeron .

Editor information

Editors and Affiliations

  1. Deptartment of Microbiology, St. Bartholomew's Hospital, Newark Street 80, London, E1 2ES, United Kingdom

    Mark Wilks

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Maheux, A.F., Bissonnette, L., Bergeron, M.G. (2013). Rapid Detection of the Escherichia coli Genospecies in Water by Conventional and Real-Time PCR. In: Wilks, M. (eds) PCR Detection of Microbial Pathogens. Methods in Molecular Biology, vol 943. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-353-4_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-60327-353-4_20

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-352-7

  • Online ISBN: 978-1-60327-353-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation