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Quartz crystal microbalance (QCM) affinity biosensor for genetically modified organisms (GMOs) detection.

A DNA piezoelectric sensor has been developed for the detection of genetically modified organisms (GMOs). Single stranded DNA (ssDNA) probes were immobilised on the sensor surface of a quartz crystal microbalance (QCM) device and the hybridisation between the immobilised probe and the target complementary sequence in solution was monitored. The probe sequences were internal to the sequence of the 35S promoter (P) and Nos terminator (T), which are inserted sequences in the genome of GMOs regulating the transgene expression. Two different probe immobilisation procedures were applied: (a) a thiol-dextran procedure and (b) a thiol-derivatised probe and blocking thiol procedure. The system has been optimised using synthetic oligonucleotides, which were then applied to samples of plasmidic and genomic DNA isolated from the pBI121 plasmid, certified reference materials (CRM), and real samples amplified by the polymerase chain reaction (PCR). The analytical parameters of the sensor have been investigated (sensitivity, reproducibility, lifetime etc.). The results obtained showed that both immobilisation procedures enabled sensitive and specific detection of GMOs, providing a useful tool for screening analysis in food samples.

Affinity Labels↗

Study design of a total reference diet for a population in Northern Italy.

A market based of 91 food items was purchased in Northern Italy, based on a list taken from a food survey previously conducted in the same area. The food items from the survey were grouped into 14 categories. Food samples were collected, homogenized, and frozen for the analysis of both the nutrient content and the levels of mineral contaminants. The study design and decision processes behind the choice of type and quantity of each food are explained. The relative quantities of each food group were compared with those from two similar studies. Finally, energy, nutrients, and mineral contaminants obtained by direct analysis were compared with the calculations given on the food composition tables. The comparison between the analyzed and calculated data only showed marked differences in phosphorus, sodium, potassium, and copper intakes.

Adult↗

Enterotoxins and phage typing of Staphylococcus aureus isolated from clinical material and food in Libya.

Enterotoxin was detected in 22 (61.1%) of the 36 S. aureus strains isolated from clinical materials and in 3 (13%) of the 23 S. aureus strains from food samples (P < 0.05). On the basis of individual types of enterotoxin, staphylococcal enterotoxin A (SEA) was produced by 11.1%, SEB by 38.9% and SEC by 22.2% of SS. aureus strains from clinical material. Of the food S. aureus strains, SEC and SED produced by 8.7% and 4.3% respectively. Of the clinical and food S. aureus strains, 52.8% and 39.1%, respectively, were typeable by the 23 phages of International Phage Set. The majority of the typeable S. aureus strains from clinical and food sources belonged to group II being at 22.2% and 17.4% respectively. Furthermore, of the 14 SEB-producing S. aureus, 42.9% were of phage group II. In conclusion, the results obtained indicate that enterotoxin-producing S. aureus strains from clinical materials in Libya are not uncommon; however, certain foods appear not to be the source of such strains. Because of the low susceptibility to bacteriophages shown by S. aureus isolated in Libya, compared to reports from several countries, other methods of typing should be used in conjunction with phage typing in epidemiological investigations concerning this organism.

Antigens, Bacterial↗

[Microbiological study of foods prepared in high-risk collective dining rooms].

BACKGROUND: Evaluation of the results of a microbiological analysis of food prepared in high-risk communal dining-rooms, in order to establish the degree of contamination, analyze the causes of this contamination, and improve the health standards of these establishments. METHODS: Descriptive, observational study with data obtained from a health inspection on 44 high-risk communal dining-rooms, which included the microbiological analysis of 90 foods, as well as the health inspection of the establishments. RESULTS: In schools mesophil micro-organisms were the most frequent contaminants; in kindergardens and homes for the elderly deficient hygiene standards were predominant in the handling of food. A greater proportion of mesophil micro-organisms were to be found during the cold months than in warmer months. Deficient hygiene indicators appeared generally in the case of food prepared in establishments in which deficiencies were observed. Psychrotrophes were not found in any of the food samples collected in kindergartens but were found in schools and homes for the elderly. CONCLUSIONS: This study indicates which problems predominate in each type of high-risk communal dining-room. Mesophils appeared in food prepared in large-scale kitchens, deficient hygiene standards were found to be associated to handling of food by non-professional staff and substandard establishments, and psychrotrophes were detected in establishments which kept left-overs. Recommendations were made as to how to eliminate the problems detected.

Food Microbiology↗

Measurements of radioactivity in environmental samples from the southern Urals.

A region between Chelyabinsk and Ekaterinburg in the Southern Urals has been heavily contaminated due to operational and accidental releases from the first Soviet plutonium production facility Mayak. In 1992 and 1993, the German Federal Office for Radiation Protection organized a measuring campaign involving two Russian institutes to assist with the validation of former Soviet measurement data. The results of this measuring campaign are reported here. Environmental samples were collected from areas affected by significant radioactive releases into the Techa river, which started in 1948, and by fallout from the explosion of a fission product storage tank in 1957. Soil, sediment, water, milk and food samples were independently analysed for 90Sr, 137Cs and plutonium by the three institutes involved. This paper presents data on the present levels of environmental radioactivity. The highest contamination of areas accessible to the local population was found in the vicinity of the Techa river around Muslumovo. Activity concentration of floodplain samples reached up to 37,000 Bq.kg-1 137Cs, 5,600 Bq.kg-1 90Sr and 9.9 Bq.kg-1 Pu. Milk and potatoes from private farms in Muslumovo showed low activity in the range from 0.7 Bq.kg-1 to 25 Bq.kg-1 90Sr. The results of the three independent measurement teams showed sufficient agreement. One Russian laboratory obtained plutonium activities that exceeded the results of the other laboratories by about 20%. Contrary to the International Chernobyl Project, there was no overestimation of 90Sr activities in the Russian analyses. Therefore, the validity of earlier data sets acquired with same methodology and quality control can be considered a valuable basis for further assessments and for dose reconstruction in epidemiological projects.

Animals↗

Chromatographic analysis of tocol-derived lipid antioxidants.

This paper provides a comprehensive overview of existing chromatographic methods for the analysis of tocol-derived lipid antioxidants in various sample matrices. After a brief introductory discussion on biological and nutritional aspects of the vitamin E active compounds, the review focuses on various techniques for the isolation, purification, chromatographic separation, and detection of tocopherols and tocotrienols. Compiled published normal-phase (NP) and reversed-phase (RP) high-performance liquid chromatographic (HPLC) methods demonstrate general trends and analytical variability and versatility of HPLC methodology. The relative merits of the two HPLC methods are assessed. NP and RP elution characteristics are delineated to aid in the identification of antioxidant components. Technical novelty of certain analytical procedures for non-food samples warrants their inclusion in this review in light of the potential applicability in food assays.

Antioxidants↗

Quantitative analysis of polybrominated diphenyl ethers in adipose tissue, human serum and foodstuff samples by gas chromatography with ion trap tandem mass spectrometry and isotope dilution.

A method based on gas chromatography (GC) separation followed by ion trap tandem mass spectrometry detection in EI mode (ITD-MS/MS), using isotope dilution, was developed for the determination of ten native polybrominated diphenyl ethers (PBDEs) and four (13)C(12)-labeled congeners in biological (fat tissue and human serum) and food samples. The highest-mass fragment ions were used as precursor ions for those congeners with molecular ions with m/z values higher than the maximum of the instrument. In these cases (hepta-BDEs and (13)C(12)-hexa-BDEs) no fragmentation was achieved under the experimental conditions employed. Repeatability (lower than 9%) and reproducibility (lower than 13%), expressed as relative standard deviation (RSD, n = 3 and 4, respectively), were satisfactory. Similarly, the coefficient of variation (n = 4) of the isotopic ratio between the two most abundant product ions was lower than 10 and 6% for native and labeled congeners, respectively. To evaluate the feasibility of the method, the optimized isotope dilution GC/ITD-MS/MS method was used for the quantitation of selected PBDE congeners in different samples including adipose tissue, human serum and foodstuff samples, from three inter-laboratory comparative exercises, covering a wide range of concentrations. A solid-phase extraction procedure, previously developed for PCB determination in small-size bird serum samples, was successfully applied to quantification of PBDEs in 1 mL samples of human serum.

Adipose Tissue↗

Patterns of PCDDs and PCDFs in human milk and food and their characterization by artificial neural networks.

Artificial neural network (ANN) has been recently introduced as a tool for data analysis. In this study, Kohonen's self-organizing maps (SOMs), a special type of neural network, were applied to a set of PCDD/PCDF concentrations found in 54 human milk and 83 food samples, which were collected in a number of countries all over the world. Data were obtained from the scientific literature. The purpose of the study was to find a potential relationship between PCDD/PCDF congener profiles in human milk and the dietary habits of the different countries in which samples were collected. The comparison of the SOM component planes for human milk and foodstuffs indicates that those countries with a greater fish consumption show also higher PCDD/PCDF concentrations in human milk. SOMs enable both the visualization of sample units and the visualization of congener distribution.

Algorithms↗

Detection of 4-oxo-2-hexenal, a novel mutagenic product of lipid peroxidation, in human diet and cooking vapor.

Since the diet plays an important role in the development of human cancer, it is important to identify mutagens in foods. We have detected a novel mutagenic product, 4-oxo-2-hexenal (4-OHE), in a model lipid peroxidation reaction mixture [H. Kasai, M. Maekawa, K. Kawai, K. Hachisuka, Y. Takahashi, H. Nakamura, R. Sawa, S. Matsui, T. Matsuda, 4-Oxo-2-hexenal, a mutagen formed by omega-3 fat peroxidation, causes DNA adduct formation in mouse organs, Ind. Health 43 (2005) 699-701]. In the present study, the contents of 4-OHE in various food samples were determined by a GC/MS method. Commercial perilla oil (derived from the seed of Perilla frutescens var. frutescens), which is rich in linolenic acid triglyceride (TG), the edible part of broiled fish, and various fried foods contained 4-OHE in the range of 1-70 microg/g. Furthermore, from the ethyl acetate trap (extracts) of the smoke released during the broiling of fish, 4-OHE was also detected by GC/MS. These results provide a warning to humans, who may be exposed to this mutagen. The 4-OHE may be produced from omega-3 polyunsaturated fats, such as alpha-linolenic acid-, docosahexaenoic acid (DHA)- and eicosapentaenoic acid (EPA)-TG, which are more easily oxidized than omega-6 fats, such as linoleic acid-TG.

Aldehydes↗

New reversed-phase liquid chromatographic method to detect aflatoxins in food and feed with cyclodextrins as fluorescence enhancers added to the eluent.

The effect of succynil-beta-cyclodextrin (beta-CD-Su), dimethyl-beta-cyclodextrin (DIMEB) and beta-cyclodextrin (beta-CD) on the fluorescence of aflatoxins B1, B2, G1, G2 and M1 (AFB1, AFB2, AFG1, AFG2 and AFM1) was studied: beta-CD-Su promoted the largest fluorescence enhancement for AFB1 and AFM1 while DIMEB showed better results for AFG1 . On the basis of the fluorescence enhancement, a new RP-HPLC method for detecting aflatoxins B1, B2, G1, G2 and M1 was developed using cyclodextrins directly dissolved in the LC eluent. Aflatoxins B1, B2, G1 and G2 were resolved using a MICRA NPS ODS-1 column using methanol-water as mobile phase to which 6 x 10(-3) M beta-CD-Su or beta-CD were added. Chromatographic responses of AFB1 and AFG1 achieved using beta-CD dissolved in the mobile phase were enhanced, respectively, 8 and 12 times, and 10 and 15 times with beta-CD-Su. Detection limits lower than 0.3 microg/kg were achieved for all the four aflatoxins. Aflatoxin M1 was analysed using a Spherisorb S3 ODS-2 Narrow Bore column and methanol-water as mobile phase with added 2 x 10(-3) M beta-CD-Su. An area enhancement of 1.5 was detected for the toxin and the detection limit achieved under these analytical conditions was lower than 0.0005 microg/kg. Both methods were statistically validated showing a linear response for all the aflatoxins tested (R2 > or = 0.99), and applied to the analysis of spiked and naturally contaminated food samples.

Aflatoxins↗

Rapid and definitive detection of Salmonella in foods by PCR.

The BAX system for screening Salmonella is one of the first commercial PCR-based systems for the detection of food-borne pathogens. It is able to give a confirmed result within 28 h. There was 98.6% and 95.8% agreement between the BAX system and conventional cultural analysis for the detection of Salmonella in artificially inoculated and uninoculated food samples, respectively. In both cases, the BAX system generated more positive detections than cultural analysis. The speed of assay, case of use and high specificity and sensitivity of the BAX system for the detection of food-borne Salmonella make it an attractive method for routine food microbiology laboratories.

Bacterial Typing Techniques↗

Use of the polymerase chain reaction and oligonucleotide probes for the rapid detection and identification of Carnobacterium species from meat.

The polymerase chain reaction (PCR) was used selectively to amplify specific rDNA sequences of Carnobacterium divergens, C. mobile, C. piscicola and C. gallinarum in purified DNA extracts, crude cell lysates and food samples. The PCR products were visualized by agarose gel electrophoresis and identified, at species level, by hybridization reactions with three specific oligonucleotide probes for C. divergens, C. mobile and C. piscicola/C. gallinarum designed from 16S rRNA sequence data. The PCR was sufficiently sensitive to amplify DNA from a single bacterium to detectable levels after 30 cycles of amplification. Both radioactive (32P) and non-radioactive alkaline phosphatase labelled probes was able to detect the PCR products. Detection was highly specific and the probes did not hybridize with DNA samples from any other of the bacterial species tested. These methods enabled the rapid and specific detection and identification of carnobacteria from pure cultures and samples of meat.

Base Sequence↗

Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction.

Five oligonucleotide sequences are described that were used as primers in the polymerase chain reaction (PCR) to amplify specific sequences from Listeria DNA. When all five primers were used in combination, three PCR products were possible; a Listeria specific product that occurs with DNA from any Listeria sp., a Listeria monocytogenes specific product that occurs only in the presence of DNA from this organism and a universal product that is found using DNA from any bacterial source. The occurrence of these PCR products was used as a diagnostic test on bacteria isolated from various food samples to detect Listeria sp. and L. monocytogenes.

Base Sequence↗

Molecular subtyping of Vibrio cholerae O1 strains recently isolated from patient, food and environmental samples in Spain.

Nineteen Vibrio cholerae O1 strains isolated in Spain from patient, food and environmental samples in the period 1990-1992 were characterized by detection of cholera toxin by enzyme immunoassay, detection of cholera toxin gene by polymerase chain reaction, and by biotyping, ribotyping and pulsed-field gel electrophoresis. Ten isolates were toxigenic and were further characterized by multilocus enzyme electrophoresis. Molecular subtyping methods allowed precise differentiation between isolates, indicating their geographic origin. Isolates associated with the ongoing seventh pandemic were distinguishable from those associated with the present Latin American epidemic. All isolates from the environment and seafood were nontoxigenic, and were genetically different and more diverse than toxigenic isolates. The data suggest that a focus of endemic cholera does not exist in Spain, and that the analyzed nontoxigenic Vibrio cholerae O1 isolates from imported seafood were not a threat to public health.

Animals↗

Direct detection of thermotolerant campylobacters in chicken products by PCR and in situ hybridization.

We have evaluated the use of PCR and fluorescent in situ hybridization (FISH) techniques for the detection of thermotolerant campylobacters in naturally contaminated chicken products. 16S rRNA sequence data was used to design two specific primers and an oligonucleotide probe for PCR and FISH analyses, respectively. The PCR protocol amplified a 439-bp fragment corresponding to a portion of specific 16S RNA gene from thermotolerant campylobacters. The detection range of the PCR assay varied between 10 cells (after enrichment) to 10(2) cells per mL (without enrichment). FISH probes were able to identify thermotolerant Campylobacter species in 'spiked' and 'unspiked' naturally contaminated samples. PCR and FISH were performed on naturally contaminated samples and compared with the isolation of cells on selective media. The in situ hybridization technique was less sensitive than PCR, although its sensitivity of detection was increased considerably after 22 h of enrichment. These results confirm the usefulness of 16S rRNA-based techniques for the direct detection of campylobacters in food samples.

Animals↗

Evaluation of reversed-phase columns for the analysis of heterocyclic aromatic amines by liquid chromatography-electrospray mass spectrometry.

Liquid chromatography coupled to mass spectrometry (LC-MS), especially by the use of electrospray ionisation source (ESI), is currently used for the analysis of heterocyclic aromatic amines (HAs) in complex samples. The present paper describes the study of the performance of different narrow-bore reversed-phase columns to achieve the best chromatographic separation for the determination of 16 HAs by LC-ESI-MS in food samples. Different parameters such as peak symmetry, resolution and number of theoretical plates have been evaluated for each column, using different chromatographic conditions. The column that provided the best results was TSK Gel Semi-Micro ODS-80TS of Tosohaas. Quality parameters have been established, obtaining good short-term precision in all cases (relative standard deviation (R.S.D.) lower than 7.7%) and low limits of detection (<13 pg injected in MS and <16 pg injected in MS/MS). The content of HAs in two beef extracts have been determined.

Amines↗

Detection and enumeration of virulent Yersinia enterocolitica in food by DNA colony hybridization.

A portion of a 44-megadalton plasmid found in Yersinia enterocolitica 8081 was used as a genetic probe to differentiate virulent and nonvirulent strains of the species. A DNA colony hybridization technique was employed. Three BamHI restriction endonuclease fragments labeled with 32P by nick translation were hybridized to lysed colonies of pure cultures, mixtures of virulent and nonvirulent cells, and portions of a food sample artificially contaminated with virulent Y. enterocolitica. The results of the colony hybridization test for virulence were the same as those obtained by the autoagglutination and suckling mouse tests. DNA colony hybridization detects pathogenic Y. enterocolitica in foods without the need for enrichment or pure cultures.

Animals↗

Assessment of heat resistance of bacterial spores from food product isolates by fluorescence monitoring of dipicolinic acid release.

This study is aimed at the development and application of a convenient and rapid optical assay to monitor the wet-heat resistance of bacterial endospores occurring in food samples. We tested the feasibility of measuring the release of the abundant spore component dipicolinic acid (DPA) as a probe for heat inactivation. Spores were isolated from the laboratory type strain Bacillus subtilis 168 and from two food product isolates, Bacillus subtilis A163 and Bacillus sporothermodurans IC4. Spores from the lab strain appeared much less heat resistant than those from the two food product isolates. The decimal reduction times (D values) for spores from strains 168, A163, and IC4 recovered on Trypticase soy agar were 1.4, 0.7, and 0.3 min at 105 degrees C, 120 degrees C, and 131 degrees C, respectively. The estimated Z values were 6.3 degrees C, 6.1 degrees C, and 9.7 degrees C, respectively. The extent of DPA release from the three spore crops was monitored as a function of incubation time and temperature. DPA concentrations were determined by measuring the emission at 545 nm of the fluorescent terbium-DPA complex in a microtiter plate fluorometer. We defined spore heat resistance as the critical DPA release temperature (Tc), the temperature at which half the DPA content has been released within a fixed incubation time. We found Tc values for spores from Bacillus strains 168, A163, and IC4 of 108 degrees C, 121 degrees C, and 131 degrees C, respectively. On the basis of these observations, we developed a quantitative model that describes the time and temperature dependence of the experimentally determined extent of DPA release and spore inactivation. The model predicts a DPA release rate profile for each inactivated spore. In addition, it uncovers remarkable differences in the values for the temperature dependence parameters for the rate of spore inactivation, DPA release duration, and DPA release delay.

Bacillus↗