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Use of the duplex TaqMan PCR system for detection of Shiga-like toxin-producing Escherichia coli O157.

Real-time PCR assays have been applied for the detection and quantification of pathogens in recent years. In this study two combinations of primers and fluorescent probes were designed according to the sequences of the rfb(Escherichia coli O157) and stx2 genes. Analysis of 217 bacterial strains demonstrated that the duplex real-time PCR assay successfully distinguished the Escherichia coli O157 serotype from non-E. coli O157 serotypes and that it provided an accurate means of profiling the genes encoding O antigen and Shiga-like toxin 2. On the other hand, bacterial strains that lacked these genes were not detected by this assay. The quantitative ranges of the real-time PCR assay for these two genes were linear for DNA concentrations ranging from 10(3) to 10(9) CFU/ml of E. coli O157:H7 in pure culture and milk samples. The real-time PCR allowed the construction of standard curves that facilitated the quantification of E. coli O157:H7 in feces and apple juice samples. The detection sensitivity of the real-time PCR assay ranged from 10(4) to 10(9) CFU/g (or 10(4) to 10(9) CFU/ml) for feces and apple juice and 10(5) to 10(9) CFU/g for the beef sample without enrichment. After enrichment of the food samples in a modified tryptic soy broth, the detection range was from 10(0) to 10(3) CFU/ml. The real-time PCR assays for rfb(E. coli) (O157) and stx2 proved to be rapid tests for the detection of E. coli O157 in food matrices and could also be used for the quantification of E. coli O157 in foods or fecal samples.

Adult↗

An evaluation of a commercially available enzyme immunoassay test for the rapid detection of salmonellae in food and environmental samples.

A total of 91 food and environmental samples were examined for the presence of salmonellae using a commercially available enzyme immunoassay kit (EIA) and a conventional culture technique. A 78% agreement was obtained, but reexamination of culture-negative, EIA-positive samples gave agreement of 86%. The problem of comparing EIA and culture results is discussed. A partially selective pre-enrichment broth was tested in 37 samples and gave better EIA ratios. Artificially contaminated cooked foods gave 100% agreement.

Culture Media↗

Comprehensive two-dimensional gas chromatography of complex samples by using a 'reversed-type' column combination: application to food analysis.

The practicability and potential of a non-orthogonal approach in comprehensive two-dimensional gas chromatography (GC x GC) were studied and compared to those of the orthogonal approach for two different complex matrices, and using conventional flame ionisation (FID) and time-of-flight mass spectrometry (ToF MS) detection. The separation of a diesel oil showed that the non-orthogonal approach also provides interesting, but completely reversed, ordered structures. For the more extensively studied flavour analysis in food samples, improved peak shapes and, also, different types of ordered structures and retention behaviour, and improved detectability for polar compounds make the two approaches complementary to each other. As a consequence, identification and/or determination of targets and/or unknowns can be performed more reliably. Analytical performance (close to three-order linearity; LODs, 2-30 pg injected in most cases; R.S.D.s, 1-6% (n = 6)) was fully satisfactory.

Chromatography, Gas↗

Aflatoxin levels in maize and maize products during the 2004 food poisoning outbreak in Eastern Province of Kenya.

BACKGROUND: On 10th May, 2004 food samples suspected to have caused acute poisoning in Makueni district were received at the National Public Health Laboratory Services (NPHLS). On analysis, they were found to be highly contaminated with aflatoxin B1. More cases of poisoning were reported in the district and in neighbouring districts of Kitui, Machakos and Thika. As at 20th July, 2004 the Ministry of Health was aware of 317 cases of which 125 resulted in deaths. OBJECTIVE: To assess the magnitude of aflatoxin contamination of maize and maize products in the affected areas. DESIGN: Random environmental sampling of maize and maize products and case/control samples of the same in the affected regions and subsequent determination of aflatoxin levels using immunoaffinity coupled with solution fluorometry. SETTING: National Public Health Laboratory Services, Ministry of Health, Kenya, from May to August, 2004. SUBJECTS: A total of 480 samples comprising 362 random environmental samples, 26 cases and 92 controls were collected and analysed. The foods analysed included maize grains, maize flour and dehulled dry maize, traditionally known as muthokoi. RESULTS: Forty six point four per cent of the environmental samples, 15% of cases and 29.3% of controls were within the maximum permissible limit of 20 microg implying that over 50% (54.4) of the total did not comply and would be regarded as unfit for human consumption. 6.9% of the environmental samples, 57.7% of cases and 21.7 of controls had levels beyond 1000 microg/Kg. The amount of aflatoxin observed in the food samples had a range of 0-58,000 microg/Kg. CONCLUSION: The population in the affected region was exposed to high levels of aflatoxin. There is need to address the issue of pre and post harvest handling of grain and establishing monitoring and surveillance for early detection and intervention.

Acute Disease↗

Development and evaluation of a 24 well microtitre plate method for isolation of Listeria spp. or Listeria monocytogenes from foods.

A rapid and simple method (24M) using 24 well microtitre plates was developed to determine the presence of Listeria monocytogenes or Listeria spp. in food samples. The 24M was composed of two 24 well microtitre plates connected with a yellow tip. The 24M was evaluated with pathogen cocktails and ground beef samples and compared with the conventional method for presumptive identification of Listeria spp. Only food-borne pathogen cocktails and ground beef samples containing L. monocytogenes or Listeria spp. showed a positive reaction in 24M after 24 h incubation at 35 degrees C. Test results were the same with the conventional method and the 24M method and showed high efficiency for recovery of Listeria spp. from foods. This new, convenient and economical method can isolate Listeria spp. simultaneously from 24 different food samples.

Animals↗

Dioxin, dibenzofuran, and polychlorinated biphenyl (PCB) levels in food from Agent Orange-sprayed and nonsprayed areas of Laos.

During the Vietnam War, a phenoxy-herbicide mixture called Agent Orange, which was contaminated with 2,3,7,8-tetrachlorodibenzodioxin, or TCDD, was used primarily for destruction of forest and other foliage in order to prevent enemy troop movement and protect American and allied troops and military bases in the south of Vietnam. Smaller areas of Laos and Cambodia were also sprayed with Agent Orange between 1962 and 1971 from fixed-wing aircraft. In 2001, 28 food samples consisting of meat, fish, and dairy products were collected in sprayed and nonsprayed areas of Laos and analyzed for dioxins, dioxinlike dibenzofurans, and selected polychlorinated biphenyl (PCB) congeners by high-resolution gas chromatography/high-resolution mass spectrometry at laboratories in Germany and Canada. Low levels of dioxin and other dioxinlike substances were found in Laotian food, similar to levels present in recent exported Vietnamese food to United States. Vietnam is geographically adjacent to Laos. Total dioxin toxic equivalent (TEQ) levels were similar in samples from sprayed and non-sprayed areas, ranging from 0.009 to 0.851 pg/g or parts per trillion (ppt) in sprayed Sepone, and from 0.022 to 0.537 pg/g or ppt wet weight in non sprayed Vientiane. However, the Laotian fish samples from the Agent Orange-sprayed area had, on average, a higher proportion of total TEQ from TCDD (31.7% vs. 9.4%) compared to the nonsprayed area. Some other food items, duck eggs and pork liver from Sepone, also had severalfold higher TCDD levels than similar food samples from Vientiane, 0.029 vs. 0.011 pg/g and 0.070 vs. 0.004 pg/g wet weight, respectively. There were no substantial differences in levels of dibenzofuran and PCB congeners. In general, elevated TCDD levels were not found in Laotian food samples. It is possible that dioxin-contaminated areas, or "hot spots," exist in Laos as they do in Vietnam, although they have not yet been identified.

2,4,5-Trichlorophenoxyacetic Acid↗

[Relationship between the toxigenicity and ribotype distribution of Pseudomonas cocovenenans subsp. farinofermentans].

By rDNA fingerprinting technique, ribotype discrimination analysis was conducted for exploring the relationship between the toxigenicity and ribotype distribution of 42 strains of Pseudomonas cocovenenans subsp. farinofermentans. The results showed that 73.8 percent of the strains were toxigenic, which were distributed within the most ribotype and isolated from either food poisoning samples or normal food samples. The other strains were not toxigenic; which were isolated from normal food samples and distributed only within a few ribotypes. Since the most clones of P. cocovenenans subsp. farinofermentans were toxigenic, their potential hazard should be concerned.

Animals↗

Monitoring of polychlorinated dibenzo-p-dioxins and dibenzofurans, dioxin-like PCBs and polycyclic aromatic hydrocarbons in food and feed samples from Ismailia city, Egypt.

Concentrations of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyl (PCBs) and polycyclic aromatic hydrocarbons (PAHs) have been determined using GC/HRMS in food (butter, seafood and meat) and feed samples (chicken, cattle and fish) purchased from Ismailia city, Egypt. PCDD/F concentrations in food samples ranged between 0.12 and 3.35 pg WHO TEQ/g wet w, while those in feed samples were between 0.08 and 0.2 pg WHO TEQ/g dry w. Levels of PCB TEQ ranged from 0.14 to 3.2 pg/g wet w in the food samples. The feed samples have an average of 0.35 pg PCB TEQ /g dry w. In this study, butter samples showed the highest contamination levels of PCDD/Fs and PCBs. The PCBs contribution to the total TEQ was on average 63% in seafood and on average 49% for meat and butter. The highest contamination levels of PCDD/Fs and PCBs were found in butter samples. The butter TEQ content is several times higher than that reported in all EU countries and exceeded the EU limits, while the PCDD/F levels in seafood and the feed samples is far below the current EU limit. Generally, congener profiles in the food samples reflect the non-industrialized nature of the city and suggest solid waste burning as a significant source of emission. Nevertheless, the profiles for butter suggest an impact from various sources. In the case of the sum of 16 PAH contamination levels in food samples were in the range of 11.7-154.3 ng/g wet w and feed samples had a range of 116-393 ng/g dry w. Benzo(a)pyrene (BaP) has been detected in the range of 0.05-3.29 ng/g wet w in the food samples; butter showed the highest contamination which exceeded the EU standard set for fats and oil. Fingerprints of PAHs suggested both petrogenic and pyrolytic sources of contamination.

Animal Feed↗

[Monitoring and official control of pesticide residues in foodstuffs in Poland in 2004].

Poland, as a Member State of the European Union, is obliged to establish efficient system of food control allowing assessment of health quality of food products for general population as well as for high risk groups, like infants and toddlers. In case of pesticide residues this task is realized by carrying out programs of monitoring and official control of foodstuffs in order to check compliance with Maximum Residue Levels set up by Polish Regulation of Minister of Health on maximum residue levels of pesticides in or on food. The Chief Sanitary Inspectorate is an authority responsible for above mentioned activities with essential participation of Department of Environmental Toxicology of National Institute of Hygiene. The analysis of food samples indicated in yearly developed plans of sampling, is carried out in 16 integrated local laboratories of National Sanitary Inspection. In 2004, 868 samples have been analyzed in system of monitoring and official control of foodstuffs in Poland. The samples were taken in retail and wholesale. Among analyzed samples 67% were fresh or frozen fruit and vegetables, and 14% were baby food. The samples have been analyzed or presence of over 50 pesticides. In the year 2004 maximum residue levels have been exceeded in 14 samples (2%) of fruit, vegetables and cereals. All samples were of Polish origin, except two samples from Spain. In samples with violated MRLs the following pesticides have been found: Benomyl group, Maneb group, Endosulfan, Mecarbam, Hexachlorobenzene, and Methyl bromide. Additionally, 49 follow-up enforcement samples has been analyzed. In none of them MRLs violation have been found.

Crops, Agricultural↗

Detection of enterotoxigenic Clostridium perfringens in food and fecal samples with a duplex PCR and the slide latex agglutination test.

A duplex PCR procedure was evaluated for the detection of Clostridium perfringens in food and biological samples and for the identification of enterotoxigenic strains. This method uses two sets of primers which amplify in the same reaction two different DNA fragments simultaneously: the 283-bp C. perfringens phospholipase C gene fragment and the 426-bp enterotoxin gene fragment. Internal primers within the two primer sets confirmed the specificity of the method by DNA-DNA hybridization with the PCR products. No cross-reaction was observed with other Clostridium species or with other bacteria routinely found in food. The detection level was approximately 10(5) C. perfringens cells per g of stool or food sample. When overnight enrichment culture was used, 10 C. perfringens cells per g was detected in 57 artificially contaminated food samples. The duplex PCR is a rapid, sensitive, and reliable method for the detection and identification of enterotoxigenic C. perfringens strains in food samples. A slide latex agglutination test was also evaluated as a rapid, simple technique for the detection of C. perfringens enterotoxin in stool samples.

Clostridium perfringens↗

[Organic pollutant residues of different origins in Navarra].

An analysis was made of residues of polychlorobyphenyls and trihalomethanes through GC-ECD and of herbicides through HPLC-PAD in samples proceeding from Navarra. Polychlorobyphenyls were detected (0.30 +/- 0.05 and 0.11 +/- 0.05 microg/l) in two of the 106 water samples analysed. Sixty-six food samples were analysed, and polychlorobyphenyls were only found in 8 samples of trout (dissimilar to dioxins: 21-194 microg/kg of fat; similar to dioxins: 41-139 microg/kg of fat). Of 107 fat samples analysed, polychlorobyphenyls dissimilar to dioxins were detected in two (27 +/- 5 and 30 +/- 5 microg/kg). Out of a total of 94 feed samples analysed, polychlorobyphenyls were detected in all the samples (12) of feed for aquaculture and their raw materials; the concentration of polychlorobyphenyls dissimilar to dioxins varied by an interval of 8-247 microg/kg of fat; polychlorobyphenyls similar to dioxins, between 18 and 107 microg/kg of fat. Contamination by polychlorobyphenyls of the fish from aquaculture could be due to the feed used in these exploitations. The average of trihalomethanes in the waters of the southern zone of Navarra (44 +/- 4 microg/l) was higher than those of the middle zone (16 +/- 1 microg/l) and the mountain zone (12 +/- 1 microg/l). The concentration of 99% of the samples fulfilled the norms on halomethanes. A relation was observed between muddiness and the concentration of trihalomethanes. Herbicide (cianazine) was only detected in one of the 135 samples of water analysed, with a concentration of (0.4 +/- 0.2 microg/l) which exceeded the established limit. The use of confirmation techniques (GC-MS, HPLC-MS/MS) would make it possible to validate these results and to expand the number of compounds analysed.

Chromatography, Gas↗

Australian survey of acrylamide in carbohydrate-based foods.

A method was developed and validated for the determination of acrylamide in carbohydrate-based foods. Solid-phase extraction employing a mixed-bed anion and cation exchange cartridge in series with a C18 extraction disk was used to clean-up water extracts of food samples before analysis by liquid chromatography coupled with tandem mass spectrometry detection. The limit of detection was calculated as approximately 25 microg kg(-1) and the limit of reporting was 50 microg kg(-1). The average method recovery for 84 samples from a range of matrices reporting was 99% with a relative standard deviation of 11.2%. A survey was conducted of 112 samples of carbohydrate-based foods composited from 547 products available in the Australian market. The analytical results were used in conjunction with Australian food consumption data derived from the 1995 National Nutrition Survey (NNS) to prepare preliminary dietary exposure estimates of Australians to acrylamide through only the food groups examined. Mean dietary exposure to acrylamide resulting from consumption of the foods tested, for Australians aged 2 years and above, was estimated as 22-29 microg day(-1) (equivalent to 0.4-0.5 microg kg(-1) bodyweight day(-1)) and between 73 and 80 microg day(-1) (1.4 and 1.5 microg kg(-1) bodyweight day(-1)) for 95th percentile consumers. Young children (2-6 years) consuming acrylamide-containing foods had a higher acrylamide exposure on a per kilogram bodyweight basis (mean 1.0-1.3 microg kg(-1) bodyweight day(-1)). The estimated exposure of Australians to acrylamide is similar to that estimated for other countries.

Acrylamide↗

Daily intake assessment of saccharin, stevioside, D-sorbitol and aspartame from various processed foods in Korea.

This study was carried out to estimate the daily intakes (EDIs) of artificial sweeteners such as saccharin, stevioside, D-sorbitol and aspartame in order to evaluate the safety of the artificial sweeteners in Korea. A total of 274 food samples were selected from the foods considered to be representative sources of artificial sweeteners in the Korean diet and analysed by using HPLC with evaporative light scattering and ultraviolet detectors. In case of aspartame, the reference values were used without instrumental analysis. The EDIs of saccharin, stevioside, D-sorbitol and aspartame for average consumers were 0.028, 0.008, 4.9 and 0.14 mg kg-1 body weight day-1, respectively, and as a proportion of the acceptable daily intake (ADI) were not higher than 1% of ADI of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). For 90th percentile consumers, the EDIs of saccharin, stevioside, D-sorbitol and aspartame were 2.0, 0.20, 141 and 4.6 mg kg-1 body weight day-1, respectively, and as a proportion of the ADI, the EDIs of saccharin and aspartame were 40.7% and 11.4% of the ADI set by the JECFA, respectively. Because JECFA did not assign ADIs for stevioside and D-sorbitol, the values for these sweeteners were not compared. According to these results, the EDIs of artificial sweeteners such as saccharin and aspartame in Korea are significantly lower than ADI set by the JECFA.

Adolescent↗

Assessing food safety and associated food handling practices in street food vending.

Street vendors in the city of Bloemfontein were investigated in order to assess the microbiological quality of the food being sold as well as the level of hygiene conditions under which these food stalls operate. The food samples which were collected included beef, chicken and gravy, while surface samples were taken from the food preparation tables and the hands of the vendors. A structured questionnaire and checklist were used in interviews to determine the status of the vending sites and associated food handling practices. The overall microbiological quality of the foods served by the street vendors was found to be within acceptable safety limits, although the presence of specific microorganisms such as Escherichia coli, Staphylococcus aureus, Salmonella and yeasts is indicative of a degree of ignorance on the part of the food handlers towards proper hygienic practices.

Colony Count, Microbial↗

Usefulness of the stomacher in a microbiological regulatory laboratory.

The relative efficiency of the Waring blender, the Stomacher 400, and the Stomacher 3500 for preparing food samples for microbiological analysis was studied. Comparative aerobic plate count (APC) values were determined on 671 samples, representing 30 categories of foods. Of the 26 categories of nonfatty foods, the blender gave significantly higher geometric mean APC values than those given by the Stomacher 400 and the Stomacher 3500 in 65 and 69 percent of the categories, respectively. In a comparison of the two Stomacher models, the Stomacher 400 gave significantly higher geometric mean APC values than these given by the Stomacher 3500 in 73 percent of the food categories. Addition of Tween 80 to four categories of fatty foods at concentrations of 0.5, 1.0, and 2.0 percent did not raise the APC values given by either model of stomacher to the levels given by the Waring blender. Overall, the efficiency of both models of Stomacher, relative to the blender and to each other, was specific and depended upon the particular food being analyzed.

Bacteria↗

Predominant serovars of Salmonella in humans and foods from Thailand.

During the period from 1993 to 1996, a total of 27,497 Salmonella isolates from humans, chicken meat, ready-to-eat Thai foods and shrimps were serotyped to know the predominant serovars of Salmonella in humans and foods in Thailand. Seventy-two and 81 serovars of Salmonella were identified in human and food samples, respectively. The significance of foods as a vehicle for human salmonellosis was discussed from an epidemiological viewpoint.

Animals↗

Identification and concentration of soy phytoestrogens in commercial dog foods.

OBJECTIVE: To identify and determine the concentrations of phytoestrogens in commercial dog foods. SAMPLE POPULATION: 24 commercial dog foods, including 12 moist or dry extruded commercial dog foods that contained soybeans or soybean fractions and 12 foods without any soybean-related ingredients listed on the label. PROCEDURE: Foods were analyzed for phytoestrogen content, including 4 isoflavones (genistein, glycitein, daidzein, and biochanin A), 1 coumestan (coumestrol), and 2 lignans (secoisolariciresinol and matairesinol) by use of acid-methanol hydrolysis and high-pressure liquid chromatography with UV-absorbance detection. Phytoestrogens were identified and quantified by reference to authentic standards. RESULTS: Isoflavones, coumestans, and lignans were undetectable in diets that did not list soybean-related ingredients on the label. Only 1 of the 12 diets that included soybean or soybean fractions had undetectable concentrations of phytoestrogens and that product contained soy fiber. The major phytoestrogens were the isoflavones daidzein (24 to 615 microg/g of dry matter) and genistein (4 to 238 microg/g of dry matter). CONCLUSIONS AND CLINICAL RELEVANCE: Soybean and soybean fractions are commonly used ingredients in commercial dog foods. Dietary intake of phytoestrogens may have both beneficial and deleterious health effects. Our results indicated that certain commercial dog foods contain phytoestrogens in amounts that could have biological effects when ingested long-term.

Animal Feed↗

Selenium content of foods consumed in Otago, New Zealand.

Selenium (Se) concentrations were determined in over 600 foods sampled in Dunedin, New Zealand. Foods included those produced in the region, foods which were expected to contribute significantly to the total Se intake, breads and other wheat products which might be affected by the importation of Australian wheat, and imported vegetarian foods. Foods with the highest Se concentrations were brazil nuts, sunflower seeds and all varieties of fish and kidneys. Somewhat lower concentrations were found in liver, pork, chicken, eggs, cashew nuts, soybeans and mushrooms. Vegetables, fruits, cereals and milk products were generally low in Se with the exceptions of mushrooms, imported legumes, rice and bananas. Those foods showing the greatest difference in Se content from those of other countries were cereal products and meat foods. The effect of a number of cooking methods on Se concentrations indicated that in general, little Se was lost during cooking. The importation of a shipment of high-Se wheat from Australia in 1984 raised Se concentrations in breads and other wheat products two to four fold.

Animal Feed↗