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The trace analysis of microorganisms in real samples by combination of a filtration microcartridge and capillary isoelectric focusing

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Abstract

Trace analysis of microorganisms in real biological samples needs very sensitive methods for their detection. Most procedures for detecting and quantifying pathogens require a sample preparation step including concentrating microorganisms from large sample volumes with high and reproducible efficiency. Electromigration techniques have great potential to include the preconcentration, separation, and detection of whole cells and therefore they can rapidly indicate the presence of pathogens. The preconcentration and separation of microorganisms from real suspensions utilising a combination of filtration and capillary isoelectric focusing was developed and the possibility for its application to real samples was verified. For our experiments, spores of Monilinia species and of Penicillium expansum were selected as model bioparticles, as they cause major losses in agrosystems. The isoelectric points of the spores of M. laxa, M. fructigena, M. fruticola, and P. expansum were determined and the method was verified using real samples taken directly from infected apples. The coupling of a filtration cartridge with a separation capillary can improve the detection limit of isoelectric focusing with UV detection by at least 4 orders of magnitude. Spores of M. fructigena and of M. laxa in numbers of hundreds of particles per milliliter were detected on a visually noninfected apple surface which was cross-contaminated during handling and storage. The efficiency of preconcentration and a preliminary identification was verified by the phenotyping technique after cultivation of the spores sampled from the apple surface.

The pre-concentration and separation of spores of Monilinia species and of Penicillium expansum from the real suspensions including combination of filtration and capillary isoelectric focusing were developed and the possibility of their application to real samples was verified. The coupling of the filtration cartridge with the separation capillary can improve the detection limit of the isoelectric focusing with the UV-detection by at least four orders of magnitude.

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Acknowledgements

This work was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (no. IAAX00310701) and by the institutional research plan AVO Z40310501.

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Authors and Affiliations

  1. Institute of Analytical Chemistry of the ASCR, v.v.i., Veveří 97, 602 00, Brno, Czech Republic

    Marie Horká, Anna Kubesová & Karel Šlais

  2. Division of Diagnostics, State Phytosanitary Administration, Šlechtitelů 23, 77900, Olomouc, Czech Republic

    Jaroslav Horký & Eva Zapletalová

  3. Department of Biochemistry, Faculty of Science, Masaryk University Brno, Kotlářská 267/2, 611 37, Brno, Czech Republic

    Anna Kubesová

Authors
  1. Marie Horká
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  2. Jaroslav Horký
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  3. Anna Kubesová
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  4. Eva Zapletalová
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  5. Karel Šlais
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Correspondence to Marie Horká.

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Horká, M., Horký, J., Kubesová, A. et al. The trace analysis of microorganisms in real samples by combination of a filtration microcartridge and capillary isoelectric focusing. Anal Bioanal Chem 400, 3133–3140 (2011). https://doi.org/10.1007/s00216-011-4975-6

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  • DOI: https://doi.org/10.1007/s00216-011-4975-6

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