DETECTING LIVE AND DEAD CELLS OF PECTOBACTERIUM ATROSEPTICUM BASED ON IMMUNOMAGNETICS SEPARATION AND STAINING

W. Sledz, A. Adamowska, J. Piosik, E. Lojkowska

Abstract


An assay that combines immunomagnetic separation (IMS) and staining by the LIVE/DEAD BacLightâ„¢ Bacterial Viability Kit was developed for the rapid detection and identification of living and dead cells of the plantpathogenic bacterium Pectobacterium atrosepticum (Pba) in potato tuber tissue. In the development of this IMS/LD assay, bacterial suspensions in buffer or potato peel extract were spiked with living and dead cells of Pba and incubated with paramagnetic beads coated with Pbaspecific antibodies. Beads with Pba cells were recovered from the suspensions with a magnet and the immobilized cells were stained with a mixture of Syto9 and propidium iodide. The collected cells were analysed by fluorescence microscopy. The assay required minimal sample preparation (e.g. no wash steps were required) and could be completed within 1 h. The detection limit was 104 and 105 cells per ml for M-280 and M-450 paramagnetic beads, respectively, which is similar to that previously reported for ELISA. The specificity and sensitivity of the IMS/LD assay were similar regardless of whether Pba cells were in potato peel extracts or in buffer. The assay also allowed the differentiation of live and dead Pba cells, which is important in predicting possible losses caused by Pba to potato crops.

Keywords


blackleg;fluorescent detection;plantpathogenic bacteria;soft rot

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DOI: http://dx.doi.org/10.4454/JPP.FA.2012.050

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