Skip to main content
SpringerLink
Log in

Development of SCAR primers based on a repetitive DNA fingerprint for Escherichia coli detection

  • Microbial Systematics and Evolutionary Microbiology
  • Published:
Journal of Microbiology Aims and scope Submit manuscript

Abstract

The present study aimed to use enterobacterial repetitive intergenic consensus (ERIC) fingerprints to design SCAR primers for the detection of Escherichia coli. The E. coli strains were isolated from various water sources. The primary presumptive identification of E. coli was achieved using MacConkey agar. Nineteen isolates were selected and confirmed to be E. coli strains based on seven biochemical characteristics. ERIC-PCR with ERIC 1R and ERIC 2 primers were used to generate DNA fingerprints. ERIC-PCR DNA profiles showed variant DNA profiles among the tested E. coli strains and distinguished all E. coli strains from the other tested bacterial strains. A 350 bp band that predominated in five E. coli strains was used for the development of the species-specific SCAR primers EC-F1 and EC-R1. The primers showed good specificity for E. coli, with the exception of a single false positive reaction with Sh. flexneri DMST 4423. The primers were able to detect 50 pg and 100 CFU/ml of genomic DNA and cells of E. coli, respectively.

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

Similar content being viewed by others

References

  • Abdallah, S.A. 2005. Detection and differentiation of Escherichia coli populations from human, animal and avian feces, and different water sources. Pol. J. Environ. Stud. 14, 639–646.

    Google Scholar 

  • Brasher, C., Panicker, G., and Bej, A. 2002. Evaluation of PCR amplification-based detection of heat-killed Escherichia coli and cell-free DNA in shellfish. Mol. Biol. Today 3, 85–90.

    CAS  Google Scholar 

  • Brenner, D.J. 1984. Family I Enterobacteriaceae Rhan (1937). pp. 410–411 In Krieg, N.R. and Holt, J.G. (eds.), Bergey’s Manual of Systematic Bacteriology, vol. 1. The Williams & Williams Co., Baltimore, Maryland, USA.

    Google Scholar 

  • Brumlik, M.J., Szymajda, U., Zakowska, D., Liang, X., Redkar, R.J., and Del Vecchio, V.G. 2001. Use of long-range repetitive element polymorphism-PCR to differentiate Bacillus anthracis strains. Appl. Environ. Microbiol. 67, 3021–3028.

    Article  PubMed  CAS  Google Scholar 

  • Cherif, A., Borin, S., Rizzi, A., Ouzari, H., Boudabous, A., and Daffonchio, D. 2002. Characterization of a repetitive element polymorphism polymerase chain reaction chromosomal marker that discriminates Bacillus anthracis from related species. J. Appl. Microbiol. 93, 456–462.

    Article  PubMed  CAS  Google Scholar 

  • Chung, C.T. and Miller, R.H. 1993. Preparation and storage of competent Escherichia coli cells. Method Enzymol. 218, 621–627.

    Article  CAS  Google Scholar 

  • Daly, P., Collier, T., and Doyle, S. 2002. PCR-ELISA detection of Escherichia coli in milk. Lett. Appl. Microbiol. 34, 222–226.

    Article  PubMed  CAS  Google Scholar 

  • Delost, M.D. 1997. Introduction to Diagnostic Microbiology: A text and work book. Mosby-Year Book, Inc, USA.

    Google Scholar 

  • dos Anjos Borges, L.G., Vechia, V.D., and Corção, G. 2003. Characterisation and genetic diversity via REP-PCR of Escherichia coli isolates from polluted waters in southern Brazil. FEMS Microbiol. Ecol. 45, 173–180.

    Article  PubMed  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Heijnen, L. and Medema, G. 2009. Method for rapid detection of viable Escherichia coli in water using real-time NASBA. Water Res. 43, 3124–3132.

    Article  PubMed  CAS  Google Scholar 

  • Kanungo, S. 2009. A simplified analysis of different Escherichia coli strains by using RAPD technique. Not. Bot. Hort. Agrobot. Cluj. 37, 257–260.

    CAS  Google Scholar 

  • Khan, A.A., McCarthy, S., Wang, R., and Cerniglia, C.E. 2002. Characterization of United States outbreak isolates of Vibrio parahaemolyticus using enterobacterial repetitive intergenic consensus (ERIC) PCR and development of a rapid PCR method for detection of O3:K6 isolates. FEMS Microbiol. Lett. 206, 209–214.

    Article  PubMed  CAS  Google Scholar 

  • Kim, H.S., Song, S.K., Yoo, S.Y., Jin, D.C., Shin, H.S., Lim, C.K., Kim, M.S., Kim, J.S., Choe, S.J., and Kook, J.K. 2005. Development of strain-specific PCR primers based on a DNA probe Fu12 for the identification of Fusobacterium nucleatum subsp. nucleatum ATCC 25586T. J. Microbiol. 43, 331–336.

    PubMed  CAS  Google Scholar 

  • Kumar, A., Asha, D., Singh, S., and Khare, S. 2011. Opc gene as a specific genetic marker for human bacterial meningitis. J. Biosci. Med. 1, DOI: 10.5780/jbm2011.7.

  • Kuo, J.T., Cheng, C.Y., Huang, H.H., Tsao, C.F., and Chung, Y.C. 2010. A rapid method for the detection of representative coliforms in water samples: polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA). J. Ind. Microbiol. Biotechnol. 37, 237–244.

    Article  PubMed  CAS  Google Scholar 

  • Lawrence, J.G., Ochman, H., and Hartl, D.L. 1991. Molecular and evolutionary relationships among enteric bacteria. J. Gen. Microbiol. 137, 1911–1921.

    PubMed  CAS  Google Scholar 

  • Louws, F.J., Fulbright, D.W., Stephens, C.T., and Bruijn, F.J. 1994. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Appl. Environ. Microbiol. 60, 2286–2295.

    PubMed  CAS  Google Scholar 

  • Mahon, C.R. and Manuselis, G. 2000. Textbook of diagnostic microbiology. 2nd edition. W.B. Saunnders Company, USA.

    Google Scholar 

  • Martin, B., Humbert, O., Camara, M., Guenzi, E., Walker, J., Mitchell, T., Andrew, P., Prudhomme, M., Alloing, G., Hakenbeck, R., and et al. 1994. A highly conserved repeat DNA element located in the chromosome of Streptococcus pneumoniae. Nucleic Acids Res. 20, 3479–3483.

    Article  Google Scholar 

  • McKillip, J.L. and Drake, M. 2000. Molecular beacon Polymerase Chain Reaction detection of Escherichia coli O157:H7 in milk. J. Food Protect. 63, 855–859.

    CAS  Google Scholar 

  • Organisation for Economic Co-operation and Development (OECD). 2003. Assessing microbial safety of drinking water. W.H.O. ISBN 92-94-09946-8.

  • Paradis, S., Boissinot, M., Paquette, N., Be’ Langer, S.D., Martel, E.A., Boudreau, D.K., Picard, F.J., Ouellette, M., Roy, P.H., and Bergeron, M.G. 2005. PPhylogeny of the Enterobacteriaceae based on genes encoding elongation factor Tu and F-ATPase betasubunit. Int. J. Syst. Evol. Microbiol. 55, 2013–2025.

    Article  PubMed  CAS  Google Scholar 

  • Pérez, F., Tryland, I., Mascini, M., and Fiksdal, L. 2001. Rapid detection of Escherichia coli in water by a culture-based amperometric method. Anal. Chim. Acta 427, 149–154.

    Article  Google Scholar 

  • Sambrook, J., Fritsch, C.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York, N.Y., USA.

    Google Scholar 

  • Tsai, T.Y., Lee, W.J., Huang, Y.J., Chen, K.L., and Pan, T.M. 2006. Detection of viable enterohemorrhagic Escherichia coli O157 using the combination of immunomagnetic separation with the reverse transcription multiplex TaqMan PCR system in food and stool samples. J. Food Protect. 69, 2320–2328.

    CAS  Google Scholar 

  • Versalovic, J., Koeuth, T., and Lupski, J.R. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 19, 6823–6831.

    Article  PubMed  CAS  Google Scholar 

  • Versalovic, J., Schneider, M., de Bruijn, F.J., and Lupski, J.R. 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Method Mol. Cell Biol. 5, 25–40.

    CAS  Google Scholar 

  • Vinten, A.J.A., Douglas, J.T., Lewis, D.R., Aitken, M.N., and Fenlon, D.R. 2004. Relative risk of surface water pollution by E. coli derived from faeces of grazing animals compared to slurry application. Soil Use Manage. 20, 13–22.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, Faculty of Science, Mahasarakham University, Mahasarakham, 44150, Thailand

    Aphidech Sangdee, Sitakan Natphosuk & Adunwit Srisathan

  2. Preclinical Division, Faculty of Medicine, Mahasarakham University, Mahasarakham, 44150, Thailand

    Kusavadee Sangdee

Authors
  1. Aphidech Sangdee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sitakan Natphosuk
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Adunwit Srisathan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kusavadee Sangdee
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Aphidech Sangdee.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sangdee, A., Natphosuk, S., Srisathan, A. et al. Development of SCAR primers based on a repetitive DNA fingerprint for Escherichia coli detection. J Microbiol. 51, 31–35 (2013). https://doi.org/10.1007/s12275-013-2244-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12275-013-2244-4

Keywords

Search

Navigation