ABSTRACT
Mass spectrometry (MS) in Selected Reaction Monitoring (SRM) mode is proposed for in-depth characterisation of microorganisms in a multiplexed analysis. Within 60-80 minutes, the SRM method performs microbial identification (I), antibiotic-resistance detection (R), virulence assessment (V) and it provides epidemiological typing information (T). This SRM application is illustrated by the analysis of the human pathogen Staphylococcus aureus, demonstrating its promise for rapid characterisation of bacteria from positive blood cultures of sepsis patients.
Subject(s)
Bacteria/classification , Bacteria/drug effects , Bacterial Typing Techniques , Mass Spectrometry/methods , Bacteria/pathogenicity , Bacterial Typing Techniques/methods , Drug Resistance, Bacterial , Humans , Reproducibility of Results , Staphylococcal Infections/microbiology , Staphylococcus aureus/classification , Staphylococcus aureus/drug effects , Staphylococcus aureus/pathogenicity , Virulence/geneticsABSTRACT
According to the World Health Organization, food safety is an essential public health priority. In this context, we report a relevant proof of feasibility for the indirect specific detection of bacteria in food samples using unlabeled phage amplification coupled to ESI mass spectrometry analysis and illustrated with the model phage systems T4 and SPP1. High-resolving power mass spectrometry analysis (including bottom-up and top-down protein analysis) was used for the discovery of specific markers of phage infection. Structural components of the viral particle and nonstructural proteins encoded by the phage genome were identified. Then, targeted detection of these markers was performed on a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. E. coli at 1 × 10(5), 5 × 10(5), and 1 × 10(6) CFU/mL concentrations was successfully detected after only a 2 h infection time by monitoring phage T4 structural markers in Luria-Bertani broth, orange juice, and French bean stew ("cassoulet") matrices. Reproducible detection of nonstructural markers was also demonstrated, particularly when a high titer of input phages was required to achieve successful amplification. This strategy provides a highly time-effective and sensitive assay for bacterial detection.
Subject(s)
Bacillus subtilis/isolation & purification , Beverages/analysis , Citrus sinensis , Coliphages/genetics , Escherichia coli/isolation & purification , Meat Products/analysis , Amino Acid Sequence , Animals , Bacillus subtilis/virology , Beverages/microbiology , Escherichia coli/virology , Food Analysis , Humans , Lysogeny , Meat Products/microbiology , Molecular Sequence Data , Peptide Library , Spectrometry, Mass, Electrospray Ionization , Swine , Viral Proteins/geneticsABSTRACT
Within the framework of medical diagnosis, the main objective is the development of hydrophilic magnetic particles for the generic capture of the nucleic acids in order to enhance the sensitivity. The strategy used in this work is based on the synthesis of cationic and hydrophilic magnetic nanoparticles bearing aminodextran. The synthesis was performed using two different processes: (i) coprecipitation of the ferrous and ferric salts in the presence of an aqueous solution of aminodextran and (ii) via adsorption of aminodextran on iron oxide nanoparticles. The obtained particles are characterized and evaluated in non-specific nucleic acids extraction and amplification.
Subject(s)
Crystallization/methods , Dextrans/chemistry , Drug Carriers/chemistry , Ferrosoferric Oxide/chemistry , Molecular Biology/methods , Nanostructures/chemistry , Nanostructures/ultrastructure , Drug Compounding/methods , Macromolecular Substances/chemistry , Materials Testing , Molecular Conformation , Molecular Probe Techniques , Particle Size , Surface PropertiesABSTRACT
Submicrometer fluorescent polystyrene (PS) particles have been synthesized via miniemulsion polymerization using CdSe/ZnS core-shell quantum dots (QDs). The influence of QD concentration, QD coating (either trioctylphosphine oxide (TOPO)-coated or vinyl-functionalized), and surfactant concentration on the polymerization kinetics and the photoluminescence properties of the prepared particles has been analyzed. Polymerization kinetics were not altered by the presence of QDs, whatever their surface coating. Latexes exhibited particle sizes ranging from 100 to 350 nm, depending on surfactant concentration, and a narrow particle size distribution was obtained in all cases. The fluorescence signal of the particles increased with the number of incorporated TOPO-coated QDs. The slight red shift of the emission maximum was correlated with phase separation between PS and QDs, which occurred during the polymerization, locating the QDs in the vicinity of the particle/water interface. QD-tagged particles displayed higher fluorescence intensity with TOPO-coated QDs compared to those with the vinyl moiety. The obtained fluorescent particles open up new opportunities for a variety of applications in biotechnology.