Pavel Rauch

Institute of Chemical Technology Prague, Praha, Praha, Czech Republic

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Publications (83)80.94 Total impact

  • [Show abstract] [Hide abstract]
    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.
    Rapid Communications in Mass Spectrometry 02/2013; 27(3):409-18. · 2.51 Impact Factor
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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.
    European Food Research and Technology 01/2012; 234(6). · 1.39 Impact Factor
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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.
    Biosensors & bioelectronics 02/2011; 26(6):2828-34. · 5.43 Impact Factor
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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.
    Biosensors & bioelectronics 02/2010; 25(9):2122-8. · 5.43 Impact Factor
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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
    European Food Research and Technology 01/2010; 231(3):467-473. · 1.39 Impact Factor
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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.
    Biosensors & bioelectronics 08/2009; 25(4):753-8. · 5.43 Impact Factor
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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.
    Czech Journal of Food Sciences 03/2009; 27:S350-3. · 0.69 Impact Factor
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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%.
    Journal of Food Biochemistry 02/2007; 13(1):21 - 29. · 0.76 Impact Factor
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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.
    Journal of Food Biochemistry 02/2007; 10(3):185 - 196. · 0.76 Impact Factor
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    ABSTRACT: Vitamin B12 was determined in biological materials by three basically different methods: microbiological assay with Lactobacillus leichmannii, microbiological assay with Escherichia coli and radioassay. The method with E. coli has a relatively low sensitivity to vitamin B12 and in some cases of vitamin B12 determination in microbial materials it can be used only after a separation of the interfering substances by gel chromatography. The procedure is suitable for orientational determinations of vitamin B12 because it is very little affected by external factors. The assay with L. leichmannii is universal owing to its high specifity and sensitivity to vitamin B12. The main disadvantage of the latter procedure depends on the high requirements for a clean atmosphere which can be mained in laboratories in industrial areas only with difficulties. These limitations do not apply to the quick and sensitive radioassay. The radioassay can be used after a suitable adjustment of the working procedure for large series of analyzes of biological materials without any preliminary separational techniques. The test can be carried out in normal laboratories providing that the principles of work with radionuclides are obeyed.
    Food / Nahrung 10/2006; 26(9):803 - 810.
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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.
    European Food Research and Technology 09/2006; 223(6):821-827. · 1.39 Impact Factor
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    ABSTRACT: The content of eggs and egg white, respectively, in various foods (pastes, mayonnaises, dressings, confections containing egg white, various ready-to- cook mixtures, ice creams, omelettes, custards, pie fillings etc) was assessed by radial immunodiffusion and preferably by rocket immunoelectrophoresis. The problem of partial ovalbumin denaturation during processing of dried eggs or egg white was overcome by using these products for the calibration of the method used. The method may also be applied to assess the degree of egg white contamination in mayonnaises in order to control the yolk and egg white separation during processing.
    Journal of the Science of Food and Agriculture 09/2006; 53(2):253 - 259. · 1.88 Impact Factor
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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.
    Journal of Food Science 08/2006; 54(4):906 - 908. · 1.78 Impact Factor
  • 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.
    Journal of Food Science 08/2006; 55(1):103 - 105. · 1.78 Impact Factor
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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.
    Analytica Chimica Acta. 01/2005;
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    ABSTRACT: Two enzyme-linked immunosorbent assays (ELISA) with chemiluminescent (CL) detection for the insecticide DDT and the group of DDT-related compounds have been optimized and characterized. Both conjugate-coated ELISAs are based on monoclonal antibodies (MAbs) of different specificity and homologous protein conjugates. Effects of several physicochemical factors (ionic strength, pH, Tween-20 and Bovine serum albumin (BSA) concentrations) and solvents (methanol, ethanol, acetone and N,N'-dimethylformamide) on the performance of the assays were studied and optimized. For the DDT-selective assay, the sensitivity, estimated as the I(50) value, was 0.6 microg/l, with a linear working range between 0.1 and 2 microg/l and a limit of detection of 0.06 microg/l. For the DDT group-selective assay, the sensitivity was 0.2 microg/l, with a linear working range between 0.07 and 1 microg/l and a limit of detection of 0.04 microg/l. CL-ELISAs were four times more sensitive compared to colorimetric ELISAs. Finally, both immunoassays were applied to the detection of DDT and group of DDT-related compounds in spiked real water, soil and food samples.
    Journal of Immunological Methods 01/2004; 283(1-2):45-57. · 2.23 Impact Factor
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    ABSTRACT: Polyclonal antisera have been raised to the whey proteins α-lactalbumin [α-La] and β-lactoglobulin [β-Lg], variants A and B. These antibody preparations have been used to develop enzyme-linked immunosorbent assays (ELISAs) for each of these proteins, which had limits of detection of 13 ng/ml [α-La], 27 ng/ml [β-Lg, variant A], and 20 ng/ml [β-Lg, variant B]. The α-La ELISA did not show any cross-reaction with β-Lg, and neither of the β-Lg ELISAs showed a cross-reactivity with α-La. However, despite the almost identical sequences of variants A and B of β-Lg, the variant A ELISA had a cross-reactivity of 66% with variant B, whilst the variant B ELISA had a cross-reactivity of more than 200% with variant A. The effect of thermal treatment on the immunoreactivity of purified whey proteins was studied by ELISA and related to changes in secondary and tertiary structure determined using CD and fluorescence spectroscopy. The immunoreactivity of α-La determined by ELISA decreased on heating above 90°C, these changes coinciding with the protein denaturation as indicated by a loss of secondary structure. In contrast, the ELISA immunoreactivity of both β-Lg variants increased after heating, a change that also coincided with changes in β-Lg secondary and tertiary structure as determined by intrinsic fluorescence of the protein. Similar thermally-induced changes in whey protein immunoreactivity were observed following heat-treatment of raw milk, the immunoreactivity of α-La being reduced whilst that of the β-Lg variants increased. When used in combination these ELISAs were able to discriminate between milks which had been pasteurized or subjected to more severe heat-treatments such as sterilization and ultra heat treatments (UHT). These data demonstrate that such immunoassays have the potential to be used as quality control methods for determining the thermal history of milks.
    Food and Agricultural Immunology 06/2003; 15(2):77-91. · 0.73 Impact Factor
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    ABSTRACT: We have developed a new enzyme-linked immunosorbent assay (ELISA) that is specific to the foodborne pathogenic micro-organism Listeria monocytogenes. It is based on an antibody raised against an L. monocytogenes cell preparation optimized for extraction of internalin B. Only in a sandwich ELISA format was the protein A-purified antibody specific to L. monocytogenes. In a competitive ELISA format, the antibody recognizes other Listeria species. The sandwich ELISA shows no recognition of L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, or L. grayii. It has a minimum detectable level for L. monocytogenes of log 10 6.37 cfu ml (1 in pure culture, is reproducible, and is unaffected by the presence of high numbers (approximately log 10 8.0 cfu ml (1) of the other Listeria species. Possible reasons for the format-dependent specificity are discussed. When the ELISA was applied to milk samples inoculated with L. monocytogenes reference material (5 cfu ml (1), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of food samples.
    Food and Agricultural Immunology 01/2003; 15. · 0.73 Impact Factor
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    ABSTRACT: In this work, a correlation study of monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) and a liquid chromatography–electrospray mass spectrometric (HPLC/ESI/MS/MS) method for the determination of N-methylcarbamate insecticides carbofuran, carbaryl and methiocarb in fruit baby food is presented. The comparison of performance characteristics of the two methods was carried out by simultaneous analysis of apple–strawberry baby food (GPC purified) extracts spiked with N-methylcarbamates at six different concentration levels. Results obtained by ELISA correlated well with those obtained by LC/MS/MS, both in terms of trueness and precision. Recoveries, i.e. the ratio of the determined concentration to the known spiked concentration, were in the 60–100% range for ELISA and in the 73–104% range by LC/MS/MS with the RSDs from seven replicate analyses 3.6–23.3 and 1.7–8.2%, respectively. The influence of sample pre-treatment on the analytical performance of immunoassay method was also assessed. Using ELISA recoveries close to 90% were obtained even in crude non-purified baby food extracts. The limits of detection (LODs) by ELISA were 0.3, 0.04 and 0.02 μg/kg−1 for carbofuran, carbaryl and methiocarb, respectively, whereas using LC/MS/MS 1 μg/kg was the detection limit for all three insecticides. The results clearly indicate that the developed ELISA is suitable for the fast, quantitative and reliable determination of carbaryl, carbofuran and methiocarb in baby food even for the analysis of crude non-purified extracts.
    Analytica Chimica Acta 01/2003; · 4.39 Impact Factor
  • Blanka Králová, Pavel Rauch
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    ABSTRACT: 1. vyd Bibliogr. na konci kapitol Rozmn
    01/2001;