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Author: Hentschel, Stefanie × Type: Article ×

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    Rapid isolation and identification of pneumonia associated pathogens from sputum samples combining an innovative sample preparation strategy and array-based detection
    (Washington : American Chemical Society, 2019) Pahlow, Susanne; Lehniger, Lydia; Hentschel, Stefanie; Seise, Barbara; Braun, Sascha D.; Ehricht, Ralf; Berg, Albrecht; Popp, Jürgen; Weber, Karina
    With this study, an innovative and convenient enrichment and detection strategy for eight clinically relevant pneumonia pathogens, namely, Acinetobacter baumannii, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae is introduced. Bacteria were isolated from sputum samples with amine-modified particles exploiting pH-dependent electrostatic interactions between bacteria and the functionalized particle surface. Following this, an asymmetric polymerase chain reaction as well as subsequent stringent array-based hybridization with specific complementary capture probes were performed. Finally, results were visualized by an enzyme-induced silver nanoparticle deposition, providing stable endpoint signals and consequently an easy detection possibility. The assay was optimized using spiked samples of artificial sputum with different strains of the abovementioned bacterial species. Furthermore, actual patient sputum samples with S. pneumoniae were successfully analyzed. The presented approach offers great potential for the urgent need of a fast, specific, and reliable isolation and identification platform for important pneumonia pathogens, covering the complete process chain from sample preparation up to array-based detection within only 4 h.With this study, an innovative and convenient enrichment and detection strategy for eight clinically relevant pneumonia pathogens, namely, Acinetobacter baumannii, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae is introduced. Bacteria were isolated from sputum samples with amine-modified particles exploiting pH-dependent electrostatic interactions between bacteria and the functionalized particle surface. Following this, an asymmetric polymerase chain reaction as well as subsequent stringent array-based hybridization with specific complementary capture probes were performed. Finally, results were visualized by an enzyme-induced silver nanoparticle deposition, providing stable endpoint signals and consequently an easy detection possibility. The assay was optimized using spiked samples of artificial sputum with different strains of the abovementioned bacterial species. Furthermore, actual patient sputum samples with S. pneumoniae were successfully analyzed. The presented approach offers great potential for the urgent need of a fast, specific, and reliable isolation and identification platform for important pneumonia pathogens, covering the complete process chain from sample preparation up to array-based detection within only 4 h.
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    Chip-based duplex real-time PCR for water quality monitoring concerning Legionella pneumophila and Legionella spp.
    (Oxford [u.a.] : Wiley-Blackwell, 2020) Reuter, Cornelia; Hentschel, Stefanie; Breitenstein, Antje; Heinrich, Eileen; Aehlig, Oliver; Henkel, Thomas; Csáki, Andrea; Fritzsche, Wolfgang
    Based on biomolecular methods, rapid and selective identification of human pathogenic water organisms becomes an important issue. Legionella spp., are pathogenic water bacteria with worldwide significance. Prevalent detection methods for these microorganisms are time and/or cost intensive. We describe a detection setup and relating DNA assay. A miniaturized real-time polymerase chain reaction (real-time PCR) for direct on-line discrimination of Legionella pneumophila and Legionella spp. was established and integrated into a real-time PCR-chip-system. The PCR-chip device combines a temperature controlling unit and a fluorescence intensity measurement. It was designed to achieve rapid amplification, using an approach of real-time fluorescence read out with the intercalating dye EvaGreen® and melting curve analysis, without requiring multiple probes. The presented results exhibit reproducibility and good sensitivity, showing that the setup is suitable for robust, rapid and cost-efficient detection and monitoring of a variety of Legionella spp.in urban water samples.