Immunomagnetic separation of Listeria monocytogenes for flow cytometric determination of viable cells in liquid

Jacobsen, Charlotte Nexmann; Fremming, Christina

doi:10.1016/s0167-7012(97)00084-5

Cited by 15 publications

(5 citation statements)

References 26 publications

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“…In general, better recoveries have been reported for MicroBeads compared to bigger beads. For example, in earlier studies where MicroBeads were applied for separation and purification for Cryptosporidia oocysts and Listeria cells, recoveries between 67 and 95% were obtained (18, 19). In comparison studies where Dynabeads were used for the detection of Mycobacterium avium , Campylobacter jejuni , or Cryptococcus neoformans , recoveries were on average lower, i.e., in the range of 47–87% (14–16).…”

Section: Discussionmentioning

confidence: 99%

“…The proposed fast screening method can be applied in principle to every bacterium or parasite if specific antibodies are available that yield a strong enough signal for the detection by flow cytometry. The MicroBeads allow a fast, efficient separation even from complex matrices (18, 19). This could be of interest especially for application in food and medical microbiology, where cells have to be isolated from complex matrices before detection.…”

Section: Discussionmentioning

confidence: 99%

“…Magnetic beads of smaller size (0.05 μm), such as MicroBeads, can reduce such problems; in addition, their ratio of surface per volume is higher. Recent reports show that cells labeled with MicroBeads can be easily isolated from complex matrices such as milk, apple juice, and manure with a high recovery of 95% and that they can be directly enumerated by flow cytometry (18, 19). For several years, magnetic beads of 0.05 μm have been used in the field of medical analysis (20), especially for isolation of specific cells in blood samples, whereas only the two studies cited earlier reported the isolation of microorganisms with MicroBeads.…”

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confidence: 99%

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Rapid and quantitative detection of Legionella pneumophila applying immunomagnetic separation and flow cytometry

et al. 2010

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Legionella is a pathogenic bacterium that establishes and proliferates well in water storage and distribution systems. Worldwide it is responsible for numerous outbreaks of legionellosis, which can be fatal. Despite recent advances in molecular and immunological methods, the official, internationally accepted detection method for Legionella spp. in water samples (ISO 11371) is still based on cultivation. This method has major disadvantages such as a long assay time of 10 days and the detection of cultivable cells only. Therefore, we developed a cultivation-independent, quantitative, and fast detection method for Legionella pneumophila in water samples. It consists of four steps, starting with (1) a concentrating step, in which cells present in one litre of water are concentrated into 5 ml by filtration (pore size 0.45 lm), (2) then cells are resuspended with sterile filtered buffer and double-stained with FITC-and Alexa-conjugated Legionellaspecific antibodies, (3) subsequently, the cells are immunomagnetically caught, and (4) finally, fluorescently labeled Legionella cells were flow cytometrically detected and quantified. The efficiency of each step was tested separately. The whole method allows detection of L. pneumophila in 180 min with a detection limit of around 500 cells/l and a recovery of Legionella cells of 52.1 % out of spiked tap water. Fluorescence microscopy and flow cytometric cell-counting correlated well. ' 2010 International Society for Advancement of Cytometry Key terms drinking water; flow cytometry; immunomagnetic separation; immunodetection; Legionella spp.; microbeads LEGIONELLA pneumophila is responsible for numerous outbreaks and isolated cases of legionellosis worldwide. Legionnaires' disease is caused by inhalation of contaminated water aerosols and is lethal in 5-40% of the cases (1). Legionella spp. are found especially in aquatic biofilms, not only in nature but also in warm water plumbing systems, air conditioners, or cooling towers. Because of the negative impact on the human health, a tolerance limit for L. pneumophila in tap water was set in different countries, including Switzerland (2), to 1000 colony-forming units (CFU)/l. The official, internationally accepted detection method for Legionella spp. in water samples (ISO 11371) is based on cultivation. Specific shortcomings of this detection method are the long assay time of 10 days, the detection of colony-forming cells only and that it is highly labor-intensive. In practice, this delay is unacceptably long, and a specific and sensitive fast screening method is of much interest. A fast screening method would be of particular interest for quickly testing water installation systems in public buildings such as hospitals, nursing homes, or hotels before closing them to prevent an outbreak of legionellosis. Several attempts were made to develop alternative fast detection methods. The most frequently used approach for fast detection is based on the polymerase chain reaction (PCR). However, the presence of PCR-inhibitory compo...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Rapid and quantitative detection of Legionella pneumophila applying immunomagnetic separation and flow cytometry

et al. 2010

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“…After optimization of several parameters, recovery rates of 86 to 99% could be obtained for cells of different Listeria strains, even in the presence of an excess microbial background. This specificity is mediated by the highly specific recognition properties of the CBDs (15,20) and represents a clear advantage over antibodies, which frequently show cross-reactions with other cells (9,22,29). Important findings were that the target specificity of CBD118 and CBD500 is maintained when the proteins are immobilized on the bead surfaces and that the specific interaction between beads and cells also worked in mixed preparations of CBD118-and CBD500-coated beads.…”

Section: Discussionmentioning

confidence: 99%

“…An ideal tool for the latter appeared to be immunomagnetic separation (IMS). However, these antibody-based anti-Listeria IMS techniques (4,6,9,26,28,29) are hampered by a number of disadvantages, i.e., (i) poor recovery rates-the percentage of cells which could be separated from cell suspensions was 20% or less (9,29), (ii) inability to detect low contamination levels (22,29), and (iii) cross-reaction with nontarget bacterial cells (9,22,29). Moreover, cross-linking and agglutination of beads were frequently observed.…”

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confidence: 99%