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Identification of nosocomial infection by electrophoretic techniques
Kubesová, Anna ; Moravcová, Dana ; Tesařová, Marie ; Horká, Marie
The detection and identification of pathogens currently relies upon a very diverse range of techniques and skills, from traditional culturing and taxonomic procedures to modern molecular biology based methods. However, conventional laboratory methods are time consuming, laborious, and they may provide both false positive or negative results, especially for closely related microorganisms. In this study, we suggest capillary electrophoresis techniques for differentiation and characterization of Methicillin-resistant and Methicillin-susceptible Staphylococcus aureus.
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High pressure modification of the simple automated liquid chromatographic system for splitless nano column gradient separations
Šesták, Jozef ; Kahle, Vladislav
Our simple liquid chromatographic system for splitless gradient nanocolumn separations at backpressure up to 50 bars was modified to run analysis under the pressure up to 300 bars. Programmable syringe pump was equipped with a 250-L high-pressure syringe and mobile phase gradients were created and stored in relatively long capillary. Available linear gradients of volumes from 5 to 50 L traced by uracil are presented. Sample introduction was performed as separate step and real large volume of sample (tens of microliters) could be injected and preconcentrated on-column. Relative standard deviation of retention times and peak areas were received (RSD < 0.3 % and RSD < 5 % respectively).
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IEF and HPLC-based methods for efficient bacterial characterization
Kubesová, Anna ; Šalplachta, Jiří ; Moravcová, Dana ; Horká, Marie
Pectobacterium and Dickeya species are responsible for blackleg and tuber soft rot diseases on crop and ornamental plants. Recently, a more virulent and aggressive Dickeya type of E. chrysanthemi was described and it becomes serious problem for potato production in Europe. In this study, we suggest procedures using two commonly available laboratory techniques, gel isoelectric focusing and liquid chromatography, for differentiation and characterization of Pectobacterium and Dickeya species. We have confirmed that fingerprinting approach can be used for bacterial differentiation, which can be further used in rapid diagnosis of plant disease.
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Silica-based monolithic capillary columns modified to zwitterionic stationary phase for hydrophilic interaction liquid chromatography
Moravcová, Dana ; Planeta, Josef ; Kahle, Vladislav ; Horká, Marie ; Roth, Michal
Zwitterionic monolithic capillary columns intended for isocratic gradient hydrophilic interaction chromatography (HILIC) separations are introduced. Silica-based capillary columns (150 mm x 0.1 mm) were prepared by acidic hydrolysis of tetramethoxysilane in the presence of polyethylene glycol and urea. The modification by a 3-(trimethoxysilyl)propyl methacrylate and then by a zwitterionic [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide to HILIC stationary phase bearing sulfoalkylbetaine groups on its surface followed. Prepared columns were characterized in HILIC separation mode employing mobile phase containing 10% (v/v) of 5 mM ammonium acetate pH = 4.5 in acetonitrile. Comparison with the commercially available ZIC-HILIC® column (Merck SeQuant®) under the same separation conditions using a mixture of aromatic carboxylic acids as a sample was done on the basis of separation efficiency of tested columns as well as retention factors and peak asymmetry of individual solutes.
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Filtration microcartridge and capillary isoelectric focusing for the analysis low number of microorganisms in real samples
Kubesová, Anna ; Horká, Marie ; Šalplachta, Jiří ; Horký, J.
At present, detection and identification of microorganisms in real biological samples needs very sensitive methods for correct diagnosis and therapy in medical practice, biotechnology, agriculture, food safety and quality etc. Accordingly, it is essential to develop new cheap techniques for pathogen identification, particularly for pathogen in complex biological samples. Pre-concentration, separation, and sensitive detection of whole cells in one step are great potential and advantage for detection of pathogens in low concentration. Combination of filtration microcartridge and capillary isoelectric focusing was developed for pre-concentration and pre-separation of microorganisms from real suspensions and the possibility its application to real samples was verified.
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