Pavel Rauch

The Police Academy of the Czech Republic in Prague, Praha, Praha, Czech Republic

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Publications (94)119.49 Total impact

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    ABSTRACT: The review focuses on the latest development of the title method. Compared with conventional instrumental techniques the method is simple and fast. It can be used for qualitative and/or semiquatitative detection and should be applied in non-laboratory environment. The principles of current formats, different markers, such as gold, carbon, and latex nanoparticles as well as possible quantitative monitoring are described. The applications generally include tests for low- or high-molecular-weight compounds, such as pesticides, drugs, mycotoxins, hormones, allergens, pathogens and metabolites in food or feed, as well as veterinary, biomedical and environmental purposes.
    No preview · Article · Feb 2014 · Chemicke Listy
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    ABSTRACT: The bacterial genus Cronobacter was established quite recently, in 2008. Therefore, its systematic classification is still in progress as well as the risk assessment of Cronobacter strains. The possibility of rapid identification within the biogroup level has an essential epidemiological significance. We examined the potential of mass spectrometry to accomplish this task on species Cronobacter sakazakii comprising eight different biogroups. Members of all Cronobacter sakazakii biogroups were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using intact cells. Analyses were performed on a Biflex IV MALDI-TOF mass spectrometer in the range of 2000 to 20 000 Da in linear mode with an accelerated voltage of 19 kV. Optimal conditions for a proper identification of biogroups, such as suitable cultivation media or growth time of bacteria, were investigated. The biomarker patterns characterizing each of the Cronobacter sakazakii biogroups were obtained. The established identification protocol was applied to ten previously non-identified strains and their biogroups were successfully determined. The presented work is the first report of successful and rapid bacterial biogroup taxonomy classification using MALDI-TOF-MS that could substitute demanding biochemical testing. Copyright © 2012 John Wiley & Sons, Ltd.
    No preview · Article · Feb 2013 · Rapid Communications in Mass Spectrometry
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    ABSTRACT: Rapid and specific detection of Cronobacter spp. in powdered infant formula milk (IFM) is of great importance for health and safety reasons. In the present study, two rapid and specific methods, the immunochromatographic strip (ICT) and the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), were tested for the detection of Cronobacter spp. in IFM. IFM samples spiked by Cronobacter spp. were correctly detected as positive by both methods. These results were verified by the classical cultivation microbiological method (ISO/TS 22964:2006). All three methods were used for the analyses of 13 IMF samples from a local market with identical results. Only one IFM sample was found to be positive. Both tested methods considerably reduced the total detection time, to 24 h (ICT) and 46 h (MALDI-TOF MS), whereas the reference ISO/TS 22964:2006 method needs 140 h.
    No preview · Article · Jun 2012 · European Food Research and Technology
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    ABSTRACT: Both immunochemical methods, sandwich ELISA and immunochromatographic detection, for detection of genus Cronobacter, have been developed. Rabbit polyclonal antibodies against dead cells of Cronobacter sakazakii Cb03 (ATCC 29544) were used. In tests of bacteria cultures from microbial collection, the ELISA distinguished between all assessed Cronobacter and Enterobacter strains. The immunochromatographic technique provided false negative results for 20 % of Cronobacter strains, and 40 % false positive results for Enterobacter strains.
    No preview · Article · Mar 2011 · Chemicke Listy
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    ABSTRACT: Members of the genus Cronobacter are opportunistic pathogens formerly known as Enterobacter sakazakii, which induce severe meningitis and sepsis in neonates and infants, with a high fatality rate. In this work, a simple and rapid immunochromatographic strip test for the detection of this pathogen was developed. Following the shortened bacteria cultivation and isolation of DNA, a specific gene sequence targeting 16S rRNA from Cronobacter spp. was amplified by PCR using 5'-end labelled specific primers. The PCR product, amplicon labelled with digoxigenin on one side and biotin on the other side, was directly added to the immunochromatographic strip test, composed of nitrocellulose membrane with bound antibody against digoxigenin in the test line. The visualization was mediated by colloidal carbon conjugated to neutravidin, and the appearance of grey/black line was indicative of the presence of specific amplicon. Colour intensity of the test line in pathogen-positive assay was visually distinguishable from that of negative sample within 10 min. The visual detection limit of PCR product was 8 ng. The specificity of the developed method was confirmed by standard microbiological techniques. Whole detection procedure with the incorporated immunostrip was applied to analysis of infant formulae samples, contaminated with less than 10 cells of Cronobacter spp. per 10 g. The results from immunochromatographic test indicated the absolute agreement with those from standard microbiological methods. Moreover, the developed procedure considerably reduced the total analysis time to 16 h whereas the reference microbiological method needs 6-7 days.
    No preview · Article · Feb 2011 · Biosensors & Bioelectronics
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    ABSTRACT: A simple immunochromatographic assay for sensitive detection of insecticide carbaryl in fruit juices was developed. The test is based on inhibition format on a nitrocellulose membrane strip. The strip was separately coated with rabbit anti-swine antibody (control line) and carbaryl hapten-OVA conjugate (test line). Colour intensity on the test line was possible to recognize visually from that of negative sample within 10min, with the detection limit of 5ngmL−1. All characteristics of the visually evaluated strips were also measured quantitatively, and then the detection limit was 1.5ngmL−1. Cross-reactions with other carbamate pesticides were not found (<1%). No matrix effects were observed when fruit juices (orange, apple, pear, banana) were diluted tenfold times before analyses. The results from immunochromatographical assay were in a good agreement with those obtained by enzyme-linked immunosorbent assay as reference method. All these facts indicate the potential of the immunochromatographic strip for the quality control of fruit juices. KeywordsCarbaryl-Immunochromatographic assay-Colloidal carbon-based immunoassay-Fruit juice
    Full-text · Article · Jul 2010 · European Food Research and Technology
  • Martina Blazková · Pavel Rauch · Ladislav Fukal
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    ABSTRACT: There is increased interest in the investigation and implementation of rapid screening methods for detection of pesticide residues. This study reports development of an immunostrip test for thiabendazole detection based on indirect competitive principle using carbon particles as a label. Nitrocellulose membrane strip was coated with a thiabendazole-protein conjugate in the defined test zone. In flow of an antibody-carbon complex and thiabendazole along the strip, the intensity of black colour formed in the test line reflected the thiabendazole concentration and semi-quantitative estimation could be carried out visually. The optimized test was accomplished within 10 min and the visual detection limit was achieved 0.25 ng mL(-1) of standard sample. Moreover, immunostrip was evaluated quantitatively using scanning densitometry. Based on standard curve, the detection limit of the proposed test was as low as 0.08+/-0.03 ng mL(-1) with an IC(50) value of 0.60+/-0.08 ng mL(-1) and a linear working range of 0.11-4.13 ng mL(-1). Results of testing precision, stability, and specificity demonstrated that the assay provided a reliable performance. This immunostrip was applied to analysis of spiked fruit juices in range of 0.05-5 mg L(-1). Matrix interferences were avoided by simple dilution of samples. Both visual and instrumental evaluations indicated a good agreement with results obtained by ELISA. Recoveries from juices were from 81.9 to 123.6% and relative standard deviations ranged from 9.9 to 19.3%. The developed strip offers potential as a useful rapid and simple method for screening of thiabendazole in fruit juices at levels far below the maximum residue limits.
    No preview · Article · Feb 2010 · Biosensors & Bioelectronics
  • M. Blažková · L. Fukal · P. Rauch
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    ABSTRACT: The aim of the review is to characterize Enterobacter sakazakii alias Cronobacter sp. as one of most dangerous microbial contaminants in foods. The microbe is a pathogen causing a serious disease in premature and newborn infants, rarely in adults. Despite a low occurrence of the infections, a high mortality (up to 80 %) has been reported. A survey of methods of its detection is given. Cultivation, chromogenic/fluorogenic media, biochemical kits and molecular genetic methods are promising in Enterobacter sakazakii screening in foods.
    No preview · Article · Feb 2010 · Chemicke Listy
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    ABSTRACT: We present a new nucleic acid lateral flow immunoassay (NALFIA) for the assessment of listeria contamination. The detection procedure starts with enrichment of sample in Half Fraser broth (24h). Following isolation of DNA, a duplex PCR is performed with two labelled primer sets, one generic and directed to a specific sequence of the gene encoding 16S rRNA from Listeria spp. and the other specific and directed to a part of the prfA gene encoding the central virulence gene regulator from the food pathogen Listeria monocytogenes (3.5h). The PCR solution is directly added to the one-step assay device and the appearance of a grey/black line is indicative of the presence of specific amplicons (max 15min). In all tests performed, the method correctly identified L. monocytogenes and strains of Listeria spp. PCR material of over 20 food samples was tested by NALFIA. The method proved to be useful for the detection of L. monocytogenes in different kinds of food samples.
    Full-text · Article · Oct 2009 · European Food Research and Technology
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    ABSTRACT: A simple and rapid immunochromatographic assay for a sensitive and inexpensive monitoring of methiocarb in surface water was developed using a binding inhibition format on a membrane strip. In the assay, detection reagent consisted of anti-methiocarb antibody and colloidal carbon-labelled secondary antibody. Methiocarb-ovalbumin conjugate was immobilized in a test line of the strip as a capture reagent. Colour intensity of the test line in methiocarb-positive assay was visually distinguishable from that of negative sample within 10min. The optimized semi-quantitative method provided a visual detection limit of 0.5ngmL(-1). Cross-reactions with other carbamate pesticides were not found (<1%). Only a negligible matrix effect of surface water was recognized. In parallel analyses of spiked water samples, the assay results were in a good agreement with those of ELISA. The stability test indicated the strips could be used at least 2 months without change in performance. All characteristics of the visually evaluated assay mentioned above were verified by instrumental quantification of colour intensity in test lines. The developed immunochromatographic assay offers potential as a useful on-site screening tool for environmental analysis.
    No preview · Article · Aug 2009 · Biosensors & Bioelectronics
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    ABSTRACT: Nucleic acid lateral flow immunoassay (NALFIA) is a method combining molecular biological principle of detection with immunochemical principle of visualisation. Following isolation of DNA from the sample, a duplex PCR with two primer sets, of which one was labelled with biotin and the other with digoxigenin or fluorescein, respectively, was performed. The PCR solution and carbon particles conjugated with avidin are directly added to the nitrocellulose membrane with two test lines of immobilised antibodies specific for digoxigenin and fluorescein. The appearance of a black line indicates the presence of specific amplicon. We would like to present the NALFIA for the simultaneous detection of L. monocytogenes in particular and the genus Listeria in general, in food. Bacteria from the genus Listeria frequently contaminate a large variety of foods. Occurrence of Listeria strains in food may indicate errors in good hygienic and manufacturing practice, only L. monocytogenes is a significant human and animal pathogen responsible for the serious illness listeriosis. Conventional microbiological methods for L. monocytogenes detection are laborious and take several days to achieve a confirmed identification.
    No preview · Article · Mar 2009 · Czech Journal of Food Sciences
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    ABSTRACT: We present a new nucleic acid lateral flow immunoassay (NALFIA) for the assessment of listeria contamination. The detection procedure starts with enrichment of sample in Half Fraser broth (24 h). Following isolation of DNA, a duplex PCR is performed with two labelled primer sets, one generic and directed to a specific sequence of the gene encoding 16S rRNA from Listeria spp. and the other specific and directed to a part of the prfA gene encoding the central virulence gene regulator from the food pathogen Listeria monocytogenes (3.5 h). The PCR solution is directly added to the one-step assay device and the appearance of a grey/black line is indicative of the presence of specific amplicons (max 15 min). In all tests performed, the method correctly identified L. monocytogenes and strains of Listeria spp. PCR material of over 20 food samples was tested by NALFIA. The method proved to be useful for the detection of L. monocytogenes in different kinds of food samples
    No preview · Article · Jan 2009
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    ABSTRACT: In this work preliminary studies on application of immunochromatographic method to detection of pesticides in food samples, in particular fruit juices, are presented. The aim was to develop immunochromatographic technique on a porous membrane for rapid detection of the pesticides thiabendazole and methiocarb in fruit juices. For detection colloidal carbon conjugated with secondary antibody was used. The tests were evaluated visually. Our results show that in the fruit juices samples examined the detection limit for thiabendazole covered the MRL (maximum residue limit) for food of plant origin established by the legislative and for methiocarb the method has to be further optimised in order to decrease the detection limit below the MRL.
    No preview · Article · Jan 2009 · Czech Journal of Food Sciences
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    ABSTRACT: ABSTRACTA commercial radioassay kit for folate (Radio-chemical Centre Amersham, England), produced for clinical analysis, has been tested for folacin assay in foodstuffs. The binding capacity of porcine binder was determined for different folates and compounds interfering with the microbiological assay. Recovery of standard addition of N5-methyltetrahydrofolate to the foodstuffs extracts estimated by radioassay varied from 96 to 105%.
    No preview · Article · Feb 2007 · Journal of Food Biochemistry
  • LADISLAV FUKAL · JAN KAS · ZDENEK SOVA · PAVEL RAUCH
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    ABSTRACT: The simultaneous presence of ascorbic acid, Cu2+ions and oxygen causes irreversible ficin inactivation. The degree of inactivation is dependent on the concentration of inhibitors. Relatively higher decrease of ficin proteolytic activity was found with high molecular substrate, hide powder azure, than with a low molecular one, N-benzoyl-L-arginine-p-nitroanilide. Gel chromatography on Sephadex G-50 showed that ficin inactivation is not due to the destruction of its molecule resulting in the decrease of its molecular weight. Inactivation of ficin proteolytic activity was associated with a partial drop in ficin immunoreactivity.
    No preview · Article · Feb 2007 · Journal of Food Biochemistry
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    ABSTRACT: We have developed two types of new paddle-style dipstick dye immunoassays. The first is genus Listeria specific and the second is specific to Listeria monocytogenes. They are based respectively, on antisera raised against heat-killed L. monocytogenes cells and against internalin B crude extract, a virulence protein found only in the pathogenic L. monocytogenes. The minimum detectable level for L. monocytogenes is 2×107CFUml−1 for strain number 88/049 in pure culture. Detection is unaffected by the presence of high numbers (approximately log 8.0CFU/ml) of the other microorganisms tested. When the dipsticks were applied to milk samples inoculated with L. monocytogenes reference material (ALM92), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of milk and ice cream food samples.
    No preview · Article · Sep 2006 · European Food Research and Technology
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    ABSTRACT: The course of thermal denaturation of hen egg white at 70°C was studied in detail by fast gel chromatography on Superose 12 and by immunochemical techniques. The mechanism of thermal denaturation differed depending on the pH of the environment. The aggregation of protein at the beginning of heat treatment was typical at pH 5 while at pH 9 the breakdown to smaller fragments prevailed. The chromatographic method for the determination of thermally undamaged (native) ovalbumin correlated well with the immunochemical method.
    No preview · Article · Aug 2006 · Journal of Food Science
  • PAVEL RAUCH · IGOR HOCHEL · JAN KÁš
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    ABSTRACT: ABSTRACTA sandwich enzyme immunoassay was developed to determine the concentration of hen egg lysozyme added as an antimicrobial agent to preserve food. The method enabled determination of egg lysozyme in beverages, milk and cheeses at a concentration as low as 1 ng/mL with an accuracy of ±12%. The recovery of the method was 85–105%. Human serum albumin (1%) was the most suitable protein for suppressing nonspecific binding.
    No preview · Article · Aug 2006 · Journal of Food Science
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    ABSTRACT: In the present work, enzyme-linked immunosorbent assays (ELISAs) with chemiluminescent detection for the determination of carbofuran, carbaryl and methiocarb were developed and the analytical parameters of these assays were compared with those of ELISAs with colorimetric detection. Both were conjugate-coated formats based on identical monoclonal antibodies and homologous protein conjugates. In comparison with colorimetric ELISA, the ability of the chemiluminescent reagents to detect lower concentrations of horseradish peroxidase allowed to decrease the optimal antibody and conjugate concentrations and to reach better analytical parameters. The experimental comparison of the analytical performance of the ELISAs was carried out by analysing extracts of apple-strawberry baby food and simply diluted fruit juices, both spiked at different concentration levels with the above mentioned pesticides. Recovery values for both ELISAs were around 100% and no matrix effects were observed when fruit juices were diluted 1:20 or more. Results obtained by ELISAs correlated well, both in terms of accuracy and precision, with those obtained by a liquid chromatography–electrospray mass spectrometry (LC/ESI/MS/MS) analysis, used as reference method to validate the immunoassays results. The limits of detection reached by using the chemiluminescent assay were 0.03, 0.007 and 0.004 ng ml−1 for carbofuran, carbaryl and methiocarb, respectively.
    Full-text · Article · Jan 2005 · Analytica Chimica Acta
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    ABSTRACT: The aim of the review is to characterize Listeria monocytogenes as one of the most dangerous microbial contaminants in foods. A survey of methods for its detection is given. Molecular genetic methods and immunoassay techniques are recognized as promising methods for Listeria monocytogenes screening in food samples. A list of commercially available kits is presented.
    No preview · Article · Jan 2005 · Chemicke Listy