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Determination of viable Escherichia coli using antibody-coated paramagnetic beads with fluorescence detection

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Abstract

A rapid and convenient assay system was developed to detect viable Escherichia coli in water. The target bacteria were recovered from solution by immunomagnetic separation and incubated in tryptic soy broth with isopropyl-β-d-thiogalactopyranoside, which induces formation of β-galactosidase in viable bacteria. Lysozyme was used to lyse E. coli cells and release the β-galactosidase. β-Galactosidase converted 4-methylumbelliferyl-β-d-galactoside to 4-methylumbelliferone (4-MU), which was measured by fluorescence spectrophotometry using excitation and emission wavelengths of 355 and 460 nm, respectively. Calibration graphs of 4-MU fluorescence intensity versus E. coli concentration showed a detection range between 8 × 104 and 1.6 × 107 cfu mL−1, with a total analysis time of less than 3 h. The advantage of this method is that it detects viable cells because it is based on the activity of the enzyme intrinsic to live E. coli.

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Acknowledgments

This work was partly supported by Hacettepe University, Scientific Research Center, Ankara Turkey (Project Number: 01 02 602 009). Ismail H. Boyaci would like to thank TUBITAK (The Scientific and Technical Research Council of Turkey) for his research fellowship.

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Authors and Affiliations

  1. Department of Food Engineering, Hacettepe University, Ankara, 06532, Turkey

    F. Ceyda Dudak & İsmail H. Boyacı

  2. Department of Chemistry, University of Cincinnati, P.O. Box 210172, Cincinnati, OH, 45221-0172, USA

    Agnese Jurkevica, Mahmud Hossain, Zoraida Aquilar, H. Brian Halsall, Carl J. Seliskar & William R. Heineman

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  1. F. Ceyda Dudak
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  2. İsmail H. Boyacı
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  3. Agnese Jurkevica
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  4. Mahmud Hossain
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  5. Zoraida Aquilar
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  6. H. Brian Halsall
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  7. Carl J. Seliskar
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  8. William R. Heineman
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Correspondence to William R. Heineman.

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Dudak, F.C., Boyacı, İ.H., Jurkevica, A. et al. Determination of viable Escherichia coli using antibody-coated paramagnetic beads with fluorescence detection. Anal Bioanal Chem 393, 949–956 (2009). https://doi.org/10.1007/s00216-008-2531-9

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  • DOI: https://doi.org/10.1007/s00216-008-2531-9

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