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PubMedDate not supplied

[Integrate cell culture--PCR (ICC/PCR) in viruses researches in environmental and food samples. Note I].

We carried out an experimental work integrating cell-culture and PCR to follow the HAV replication, to verify the PCR positiveness times and to confirm infectious viral particles presence. In tests HAV strain H59-175 was used. We proceeded to an initial valuation of the lowest viral concentration detectable by PCR. With viral titres between 10(5)-10(7) PFU/ml, the highest positive dilution resulted 10(-4)-10(-5). Then the 1 log lower dilution was inoculated in cell culture. At fixed times we proceeded to take surnatant and lisate samples for PCR test. After cell culture integration, positiveness was obtained in 72-120 hours against the 10-18 days necessary for CPE appearance.

Cell Culture Techniques↗
PubMed2004

Amperometric biosensor based on denatured DNA for the study of heavy metals complexing with DNA and their determination in biological, water and food samples.

Amperometric biosensor (BS) has been elaborated based on the stationary mercury-film electrode (SMFE) with silver support and cellulose nitrate (CN) membrane containing immobilized single-stranded DNA (ssIDNA). The sorption isotherms and ssDNA-heavy metal binding constants have been obtained with the BS. According to these data, the chosen heavy metals form the following series of binding strength with ssIDNA: Pb(II)>Fe(III)>Cd(II). It has been found that upon the competitive adsorption, there exists practically simultaneous sorption of different ions at ssIDNA containing membrane. The method of the determination of heavy metals based on preconcentration of metal ions on the BS followed by the destruction of DNA-metal complexes with ethylenediamine tetraacetate (EDTA) and voltammogram recording has been proposed. The lower limits of detectable contents are 1.0x10(-10), 1.0x10(-9) and 1.0x10(-7) mol l(-1) for Pb(II), Cd(II) and Fe(III), respectively. Heavy metals have been assayed in natural and drinking water, milk and blood serum samples even under simultaneous presence with a selectivity factor of 1:10. The effect of matrix components has been estimated.

Animals↗
PubMedDate not supplied

Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/PCDF) in food samples collected in southern Mississippi, USA.

In 1994, we analyzed 43 foodstuff samples from local supermarkets in southern Mississippi, USA, for PCDD/PCDF. 2,3,7,8-Cl4DD could be quantified in 31 of these samples. On a lipid basis, levels in meat (0.53-1.10 pg I-TEQ/g) and dairy products (0.42-1.10 pg I-TEQ/g) were slightly lower than those reported from other industrialized countries. While levels in dairy samples from the United States and Europe are comparable, there is a difference in the contribution of individual congeners to the I-TEQ: for example, in milk samples from Germany approximately 40% of the I-TEQ is due to the presence of 2,3, 4,7,8-Cl5DF while in the Mississippi samples this congener only contributes 16%. The highest concentrations of PCDD/PCDF in our study were detected in the farm-raised catfish (10.2-27.8 pg I-TEQ/g). A unique finding was that in addition to the 2,3,7,8-substituted PCDD/PCDF the catfish samples contained many non-2,3,7,8-substituted congeners. This is unusual because vertebrate animals selectively eliminate or metabolize the non-2,3,7,8-substituted congeners.

Benzofurans↗
PubMedDate not supplied

Comparison of four methods for digesting food samples for determination of trace levels of cadmium by flameless atomic absorption spectrophotometry.

We compared 4 digestion procedures, namely, sulfuric-nitric acid in an open flask, nitric acid under pressure, sulfuric-nitric acid with refluxing, and nitric-hydrochloric-peroxide with refluxing, for the determination of cadmium by flameless atomic absorption spectrophotometry in 3 foodstuffs: rice, beef, and cream cheese. The foodstuffs were homogenized and divided into several batches for analysis. The results were evaluated using a 2-way cross analysis of variance. The study revealed that the digestion procedure was a highly significant factor (P less than 10(-4] in the analysis of the 3 foods; whereas the nature of the foodstuffs was not significant for rice and meat and only slightly significant (P less than 10(-2] for cream cheese. When the foodstuffs were spiked with a known amount of cadmium, we observed a loss of the metal when the sulfuric-nitric acid procedure in an open flask and the nitric-hydrochloric-peroxide digestion procedure were used. Taken together, the results of the present study indicate that the choice of the reagents used for digestion of foodstuffs is a crucial factor for cadmium determination by flameless atomic absorption spectrophotometry.

Animals↗
PubMed1994

Immunomagnetic separation as a sensitive method for isolating Escherichia coli O157 from food samples.

Minced beef samples inoculated with Escherichia coli O157 were cultured in buffered peptone water supplemented with vancomycin, cefsulodin and cefixime (BPW-VCC) and subcultured to cefixime tellurite sorbitol MacConkey (CT-SMAC) agar both directly and after immunomagnetic separation (IMS) of the organism with magnetic beads coated with an antibody against E. coli O157 (Dynabeads anti-E. coli O157, Dynal, Oslo). E. coli O157 was recovered from initial inocula of 200 organisms/g by direct subculture and 2 organisms/g by IMS. Twelve strains of E. coli O157 of different combinations of phage type, H antigen and toxin genotype were all recovered from initial inocula of two organisms/g by IMS. Non-specific binding of other organisms to the magnetic beads could be reduced by washing of the beads in PBS with Tween-20 0.002-0.005% E. coli O157 was not bound by magnetic coated with an unrelated antibody. During investigation of a dairy herd that was possibly linked to a small outbreak of infection with E. coli O157, the organism was isolated from 2 of 279 forestream milk samples from individual cattle; both isolates were made only by the IMS technique. IMS is rapid, technically simple, and a specific method for isolation of E. coli O157 and will be useful in epidemiological studies.

Animals↗
PubMed1964

ISOLATION OF SALMONELLAE FROM FOOD SAMPLES. VI. COMPARISON OF METHODS FOR THE ISOLATION OF SALMONELLA FROM EGG PRODUCTS.

THE RECOVERY OF SALMONELLAE FROM EGG PRODUCTS WAS STUDIED, BY USE OF THREE DIFFERENT ENRICHMENT PROCEDURES: (i) selenite broth, (ii) selenite broth containing 10% sterile feces, and (iii) the lactose pre-enrichment procedure. Brilliant Green Agar was used throughout as the recovery medium. Although the lactose pre-enrichment methodology promoted Salmonella recovery from samples containing small numbers of dormant organisms, the efficiency of this enrichment method is adversely affected by unfavorable coliform-Salmonella ratios. Under such conditions, early subculture of lactose broth into selenite broth is indicated. Selenite broth containing 10% sterile feces was more efficient than the lactose pre-enrichment methodology in promoting the growth of "dormant" salmonellae. Albumen adversely affected recovery of salmonellae from selenite broth, whereas whole egg and egg yolk enhanced Salmonella recovery from this medium. The selenite-feces medium presents a solution to the major problems encountered in the detection of salmonellae in egg products and offers an approach to a single medium in which food-borne salmonellae will manifest themselves with a minimum of laboratory manipulation.

Agar↗
PubMed1998

Controlled multiplex PCR of enterotoxigenic Clostridium perfringens strains in food samples.

A controlled multiplex polymerase chain reaction (PCR) for the detection of Clostridium(C.) perfringens enterotoxin gene (cpe) was established and compared with an in vitro antigenic detection method. Thecpe PCR and the classical method of electric immunodiffusion gave identical results. The predicted specific amplicon of the cpe gene was generated from all of the tested enterotoxigenic C. perfringens strains. In contrast, cultures of any other Clostridium species tested by PCR were negative (100% sensitivity, 100% specificity). Addition of an alphatoxin (plc) gene specific PCR as an in process control reaction was performed in order to prevent false negative PCR results. The PCR detection limit was 0.5 ng of genomic C. perfringens DNA per ml of bouillon culture. By contaminating minced meat with C. perfringens reference strains, the multiplex PCR was established as a tool for routine diagnostic laboratories. The detection limit was approximately 3.0x10(5)C. perfringens cells per gram meat. The results demonstrate the multiplex PCR as an easy, specific, sensitive and time saving diagnostic procedure. Application of this improved method should enhance the knowledge concerning epidemiological aspects of food borne diseases caused by enterotoxigenic C. perfringens.

Clostridium perfringens↗
PubMed1982

High-performance liquid chromatography of fat-soluble vitamins: separation and identification of vitamins D2 and D3 and their isomers in food samples in the presence of vitamin A, vitamin E and carotene.

Vitamins D2 and D3 and their corresponding previtamins and provitamins were resolved by reversed-phase high-performance liquid chromatography using a ternary solvent system (acetonitrile-methanol-water) pumped according to a gradient elution programme. The D vitamins were also resolved in the presence of other lipid-soluble vitamins (A, E and K1) and carotene. The peaks were monitored with a UV-visible variable-wavelength detector and were detected at their maximum absorbance, resulting in maximum sensitivity. Lipid-soluble vitamins and carotene were resolved in extracts obtained from oils and butter, thus permitting their identification in a single chromatographic run.

Butter↗
PubMed1991

Kinetic determination of iodide in pharmaceutical and food samples.

A kinetic method for the determination of iodide based on its inhibitory effect on the Pd(III) catalysed reaction between ethylenediaminetetraacetic acid (EDTA)-Co(III) and the hypophosphite ion is described. The reaction was followed spectrophotometrically by measuring the decrease in the absorbance at 540 nm. Under the optimum experimental conditions of 2.6 x 10(-3) mol dm(-3)Co(III)-EDTA, 0.4 mol dm(-3)H2PO2-, pH 3.2 (adjusted with Britton-Robinson buffer), 0.57 micrograms ml(-1) Pd(III) and 20 +/- 0.2 degrees C, iodide was determined in the range 2-28 ng ml(-1). The method was applied to the determination of iodide in pharmaceutical products, iodinated salts, cow's milk and infants' powdered milk.

Animals↗
PubMed2006

Rapid determination of fumonisin B1 in food samples by enzyme-linked immunosorbent assay and colloidal gold immunoassay.

A rapid enzyme-linked immunosorbent assay (ELISA) test (microwell plate) and a membrane-based colloidal gold immunoassay in flow-through and lateral-flow formats for the rapid detection of fumonisin B1 (FB1) were developed. The rapid microwell assay can be completed within 20 min with the detection limit of 0.5 +/- 0.2 microg/L. Membrane-based colloidal gold immunoassays had a visual detection limit of 1.0 microg/L for FB1 with the detection time of <10 min. Matrix interference was eliminated by 15-fold dilutions of methanol extracts with buffer. These immunoassays can be used as quantitative or qualitative tools for the rapid detection of FB1 residues in 10-20 min on-site.

Arachis↗
PubMed1961

Isolation of Salmonellae from food samples. IV. Comparison of methods of enrichment.

A comparison of various methods of enhancing frequency of Salmonella isolations revealed that inoculation of a second enrichment broth, with culture from the first, was no improvement over the single direct enrichment method. It was inferior to centrifugation. Selenite was observed to produce more positive isolations at 48 hr than at 24. No change occurred in tetrathionate. Reconstitution of dried albumen with water produced a significant increase in isolations over direct inoculation of enrichment broth in the case of tetrathionate but not selenite broth.Pre-enrichment in lactose broth before inoculation of enrichment media was vastly superior to reconstitution in water for both enrichment broths. A comparison of results obtained using dulcitol, mannitol, lactose and carbohydrate-free purple broths in pre-enrichment indicated that the carbohydrate added was immaterial.

Culture Media↗
PubMed2005

High occurrence and persistence of antibiotic-resistant enterococci in poultry food samples in Portugal.

OBJECTIVES: We determined the presence of antibiotic-resistant enterococci (ARE) in commercialized poultry samples from Portugal and analysed their clonal diversity and the resistance genes harboured by these strains. METHODS: Ninety-nine retail poultry samples of 10 widely commercialized brands were studied (1999-2001). Samples were enriched and plated on selective media with and without vancomycin, gentamicin, streptomycin or kanamycin. Antibiotic susceptibility was established following standard criteria. Identification and detection of genes coding for resistance were determined by PCR. Clonal relatedness was established by PFGE. RESULTS: A high percentage of samples contained vancomycin-resistant enterococci (VRE) (48%), or enterococci highly resistant (HLR) to gentamicin (34%), streptomycin (32%) or kanamycin (30%). Co-resistance to tetracycline, erythromycin, ciprofloxacin and quinupristin/dalfopristin was observed in most of these isolates. VRE were classified as VanA phenotype-vanA genotype (38% of samples), VanB phenotype-vanA (13%) or VanC phenotype-vanC1 (23%). All HLR to gentamicin isolates contained aac(6')-Ie-aph(2'')-Ia. We detected erm(B) in both erythromycin-resistant and -susceptible isolates. Some VRE and HLR to gentamicin strains were recovered from different samples and brands. Long-term persistence of particular VRE strains (>2 years), exhibiting different Van phenotypes, was observed. CONCLUSIONS: High occurrence of ARE suggests maintenance of selective pressure by the use of antibiotics/other substances in the Portuguese poultry environment. Persistence of a number of widespread PFGE types containing different resistance genes might reflect environmental/host-adapted enterococcal strains that might contribute to the maintenance of antibiotic resistance, thus constituting a resistance reservoir that is non-sensitive to banning interventions.

Acetyltransferases↗
PubMed2005

Analysis of acrylamide in food samples by capillary zone electrophoresis.

Conditions for the determination of acrylamide (AA) after derivatisation with 2-mercaptobenzoic acid by capillary zone electrophoresis were established. A derivatisation reagent-acrylamide ratio of 35:1 was selected as optimum and the reagent excess was not removed as it did not affect the determination of acrylamide by CZE. The best separation was achieved using a 40 mM phosphate buffer at pH 8.0, working at 25 kV in un-coated fused silica capillaries. Linear calibration curves over the range studied (0.3-100 microg mL(-1)), the limit of detection (0.07 microg mL(-1)), and both run-to-run (RSD values of 5.8 and 2.2% for concentration at low and medium concentration levels, respectively) and day-to-day precisions (up to 11.2 and 6.7% at low and medium concentration levels, respectively) were established. Finally, the applicability of the CZE proposed methodology was demonstrated by analyzing levels of acrylamide present in different foodstuff products such as home made french fries, breakfast cereals and biscuits.

Acrylamide↗
PubMed2001

Real-time detection of L-ascorbic acid and hydrogen peroxide in crude food samples employing a reversed sequential differential measuring technique of the SIRE-technology based biosensor.

Detection of the common electrochemical interferents, ascorbic acid and hydrogen peroxide, using a SIRE (Sensors based on Injection of the Recognition Element) technology based biosensor in reverse mode operation is reported. The differential measuring principle employed in the SIRE biosensor during operation in reverse mode is such that the sample is measured first in the presence of enzyme (yielding matrix signal only), and then measured again in the absence of enzyme (yielding signal from matrix+analyte). Subtraction of the signal obtained in the presence of enzyme from the signal obtained in the absence of enzyme gives a specific signal for the analyte only and correlates directly to its concentration in solution. The linear range for the determination of ascorbic acid and hydrogen peroxide was 0-3 mM and 0-2 mM, respectively, with an enzyme concentration of 25 U ascorbate oxidase/ml and 1000 U catalase/ml. The reproducibility was 5% for ascorbic acid (R.S.D. n=15) and 10% for hydrogen peroxide (R.S.D. n=18). The cost per measurement was 0.28 USD for ascorbic acid analysis and 0.0008 USD for hydrogen peroxide analysis. The degradation of ascorbic acid in cereal was followed in real-time, as was the stabilization of low pH on the degradation process.

Ascorbate Oxidase↗
PubMed2001

Enzymatic reactions for the determination of sugars in food samples using the differential pH technique.

All ATP coupled reactions, when performed at neutral or moderately alkaline pH, produce an acidification of the reaction mixture. The detection of small pH changes--0.1 mpH (1 mpH = 10(-3) pH)--in a constant buffering capacity solution makes it possible to quantify, over a wide concentration range (1-1500 mmol L(-1)), various analytes with very high precision and accuracy. Glucose, fructose, glycerol and gluconic acid can be analysed in less than 1 min with a single step reaction. Wine samples were analysed using the hexokinase reaction for glucose + fructose (sugars undergoing fermentation) and compared against an established method, showing excellent performance over the whole range of concentrations (R = 0.9994). Increased sensitivity in some applications can be obtained by cycling reactions, e.g. a kinase reaction followed by a phosphatase reaction. in a one step analysis, as required for lactulose assay in milk, a useful indicator of heat treatment damage. A sensitivity well below 0.1 mmol L(-1) in the original milk sample has been demonstrated.

Animals↗
PubMed1961

Isolation of Salmonellae from food samples. V. Determination of the method of choice for enumeration of Salmonella.

Comparison of three methods by which salmonellae may be isolated and enumerated from dried albumen, direct inoculation of enrichment media, centrifugation of samples, and pre-enrichment in noninhibitory media, reveals pre-enrichment to be the method of choice. The superiority of pre-enrichment manifests itself in replicate aliquots of the same sample by producing a statistically significant increase in numbers of isolations of salmonellae and in empirical use with various albumen samples by consistently higher values of most probable numbers (MPN). The primary factor involved in this superiority appears to be the greater ability of small numbers of salmonellae to initiate growth in the nonselective mannitol purple sugar broth than in the inhibitory enrichment media. The method of analysis recommended entails inoculation of mannitol broth pre-enrichment medium, transfer of 24-hr culture aliquots to tetrathionate broth, and streaking on brilliant green agar for isolation of salmonellae.

Agar↗