The trace analysis of microorganisms in real samples by combination of a filtration microcartridge and capillary isoelectric focusing

• Published in: Analytical and bioanalytical chemistry 2011-07, Vol.400 (9), p.3133-3140
• Main Authors: HORKA, Marie, HORKY, Jaroslav, KUBESOVA, Anna, ZAPLETALOVA, Eva, SLAIS, Karel
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Apples; Ascomycota - isolation & purification; Biochemistry; Capillarity; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Electrophoresis, Capillary - instrumentation; Electrophoresis, Capillary - methods; Equipment Design; Exact sciences and technology; Filtration; Filtration - instrumentation; Filtration - methods; Food Science; Isoelectric focusing; Isoelectric Focusing - instrumentation; Isoelectric Focusing - methods; Laboratory Medicine; Malus - microbiology; Mathematical models; Microorganisms; Monitoring/Environmental Analysis; Original Paper; Penicillium - isolation & purification; Sensitivity and Specificity; Separation; Spores; Spores, Fungal - isolation & purification
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-011-4975-6

Trace analysis of microorganisms in real biological samples needs very sensitive methods for their detection. Most procedures for detecting and quantifying pathogens require a sample preparation step including concentrating microorganisms from large sample volumes with high and reproducible efficiency. Electromigration techniques have great potential to include the preconcentration, separation, and detection of whole cells and therefore they can rapidly indicate the presence of pathogens. The preconcentration and separation of microorganisms from real suspensions utilising a combination of filtration and capillary isoelectric focusing was developed and the possibility for its application to real samples was verified. For our experiments, spores of Monilinia species and of Penicillium expansum were selected as model bioparticles, as they cause major losses in agrosystems. The isoelectric points of the spores of M. laxa , M. fructigena , M. fruticola , and P. expansum were determined and the method was verified using real samples taken directly from infected apples. The coupling of a filtration cartridge with a separation capillary can improve the detection limit of isoelectric focusing with UV detection by at least 4 orders of magnitude. Spores of M. fructigena and of M. laxa in numbers of hundreds of particles per milliliter were detected on a visually noninfected apple surface which was cross-contaminated during handling and storage. The efficiency of preconcentration and a preliminary identification was verified by the phenotyping technique after cultivation of the spores sampled from the apple surface. Figure The pre-concentration and separation of spores of Monilinia species and of Penicillium expansum from the real suspensions including combination of filtration and capillary isoelectric focusing were developed and the possibility of their application to real samples was verified. The coupling of the filtration cartridge with the separation capillary can improve the detection limit of the isoelectric focusing with the UV-detection by at least four orders of magnitude.