Genetics-based methods for detection of Salmonella spp. in foods

Genetic methods are now at the forefront of foodborne pathogen testing. The sensitivity, specificity, and inclusivity advantages offered by deoxyribonucleic acid (DNA) probe technology have driven an intense effort in methods development over the past 20 years. DNA probe-based methods for Salmonella spp. and other pathogens have progressed from time-consuming procedures involving the use of radioisotopes to simple, high throughput, automated assays. The analytical sensitivity of nucleic acid amplification technology has facilitated a reduction in analysis time by allowing enriched samples to be tested for previously undetectable quantities of analyte. This article will trace the evolution of the development of genetic methods for detection of Salmonella in foods, review the basic assay formats and their advantages and limitations, and discuss method performance characteristics and considerations for selection of methods.     

Reveal Listeria 2.0 test for detection of Listeria spp. in foods and environmental samples

A Performance Tested Method validation study was conducted for a new lateral flow immunoassay (Reveal Listeria 2.0) for detection of Listeria spp. in foods and environmental samples. Results of inclusivity testing showed that the test detects all species of Listeria, with the exception of L. grayi. In exclusivity testing conducted under nonselective growth conditions, all non-listeriae tested produced negative Reveal assay results, except for three strains of Lactobacillus spp. However, these lactobacilli are inhibited by the selective Listeria Enrichment Single Step broth enrichment medium used with the Reveal method. Six foods were tested in parallel by the Reveal method and the U.S. Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) reference culture procedure. Considering data from both internal and independent laboratory trials, overall sensitivity of the Reveal method relative to that of the FDA/BAM procedure was 101%. Four foods were tested in parallel by the Reveal method and the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) reference culture procedure. Overall sensitivity of the Reveal method relative to that of the USDA-FSIS procedure was 98.2%. There were no statistically significant differences in the number of positives obtained by the Reveal and reference culture procedures in any food trials. In testing of swab or sponge samples from four types of environmental surfaces, sensitivity of Reveal relative to that of the USDA-FSIS reference culture procedure was 127%. For two surface types, differences in the number of positives obtained by the Reveal and reference methods were statistically significant, with more positives by the Reveal method in both cases. Specificity of the Reveal assay was 100%, as there were no unconfirmed positive results obtained in any phase of the testing. Results of ruggedness experiments showed that the Reveal assay is tolerant of modest deviations in test sample volume and device incubation time.     

Validation of RAPID'Staph for enumeration of Staphylococcus aureus in foods. Performance-Tested Method

RAPID'Staph (Bio-Rad Laboratories, Hercules, CA) is a medium for differentiation and enumeration of coagulase-positive Staphylococcus aureus in food. RAPID'Staph medium is based on a Baird Parker formula optimized for the detection and enumeration of S. aureus in 24 h. The principle of the medium relies on the capacity of S. aureus to reduce tellurite (production of black colonies) and to provoke proteolysis of egg yolk (production of clear halo around the colony). Four foods (pasteurized whole milk, custard pie, processed ham, and smoked salmon) were selected to compare the performance of RAPID'Staph agar to AOAC Official Method 975.55. Method comparison studies demonstrated excellent agreement between the methods. Inclusivity and exclusivity rates of the medium were 100%. RAPID'Staph agar performed as expected when minor procedural variations were introduced, validating the ruggedness of the method. There was no difference in performance between the dehydrated and ready-to-use formulations of the media.     

Reveal Salmonella 2.0 test for detection of Salmonella spp. in foods and environmental samples. Performance Tested Method 960801

Reveal Salmonella 2.0 is an improved version of the original Reveal Salmonella lateral flow immunoassay and is applicable to the detection of Salmonella enterica serogroups A-E in a variety of food and environmental samples. A Performance Tested Method validation study was conducted to compare performance of the Reveal 2.0 method with that of the U.S. Department of Agriculture-Food Safety and Inspection Service or U.S. Food and Drug Administration/Bacteriological Analytical Manual reference culture methods for detection of Salmonella spp. in chicken carcass rinse, raw ground turkey, raw ground beef, hot dogs, raw shrimp, a ready-to-eat meal product, dry pet food, ice cream, spinach, cantaloupe, peanut butter, stainless steel surface, and sprout irrigation water. In a total of 17 trials performed internally and four trials performed in an independent laboratory, there were no statistically significant differences in performance of the Reveal 2.0 and reference culture procedures as determined by Chi-square analysis, with the exception of one trial with stainless steel surface and one trial with sprout irrigation water where there were significantly more positive results by the Reveal 2.0 method. Considering all data generated in testing food samples using enrichment procedures specifically designed for the Reveal method, overall sensitivity of the Reveal method relative to the reference culture methods was 99%. In testing environmental samples, sensitivity of the Reveal method relative to the reference culture method was 164%. For select foods, use of the Reveal test in conjunction with reference method enrichment resulted in overall sensitivity of 92%. There were no unconfirmed positive results on uninoculated control samples in any trials for specificity of 100%. In inclusivity testing, 102 different Salmonella serovars belonging to serogroups A-E were tested and 99 were consistently positive in the Reveal test. In exclusivity testing of 33 strains of non-salmonellae representing 14 genera, 32 were negative when tested with Reveal following nonselective enrichment, and the remaining strain was found to be substantially inhibited by the enrichment media used with the Reveal method. Results of ruggedness testing showed that the Reveal test produces accurate results even with substantial deviation in sample volume or device development time.     

Validation of a microwell DNA probe assay for detection of Listeria spp. in foods Performance-tested method 010403

A new DNA hybridization assay in microwell format for detection of Listeria spp. in foods and environmental samples was developed. This assay uses Listeria-specific oligonucleotide probes labeled with horseradish peroxidase and a photometrically determined end point. Validation studies with 15 different food commodities and a variety of environmental sample types were conducted to compare the performance of this alternative test versus reference methods. Meats, seafood, dairy products, and vegetables comprised the categories of food tested. Food samples were inoculated at 2 levels and refrigerated or frozen for at least 72 h. Uninoculated (negative) control samples were included in each trial. Samples were enriched according to the procedure recommended by either the U.S. Food and Drug Administration (FDA) or the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS). Samples enriched for 24 h were transferred to Oxford agar plates and incubated for 24 h. The surface of the plates was then swabbed and any growth present was transferred to phosphate buffer solution for the performance of the DNA assay. A standard confirmation procedure was used to compare the number of positive samples obtained with the DNA method versus reference methods. Statistical analyses of the results indicate that the proposed alternative method performs equally to cultural reference methods. The DNA assay is able to detect as low as 1 colony-forming unit of Listeria in a 25 g food sample, with results available as early as 48 h after the start of sample enrichment.     

Chromatographic methods for tetracycline analysis in foods.

The tetracyclines have served for decades as an important class of antibiotics in food animal health and production. As such, they have also been a source of concern for residue monitoring authorities around the world. In response to this concern a number of microbial inhibition, immunoassay and bacterial receptor methods have evolved for the detection of this class of compounds in various foods of animal origin. However, these methods often lack specificity and are subject to false positive and false negative results. For these reasons a number of chromatographic methods for the separation and determination of the tetracyclines isolated from foods have been developed that are capable of identifying and quantifying individual tetracycline drugs. We present here an overview of tetracycline analytical methods, including microbial inhibition, immunoassay and receptor technologies for detection, techniques for isolation from food matrices, and thin-layer chromatographic, high-performance liquid chromatographic, gas chromatographic and mass spectrometric procedures for determination of this class of compounds. A discussion of the variables involved in such methodology and a review of method criteria are offered.     

HPLC荧光法快速检测食品中的NaNO2

建立了HPLC荧光检测法测定不同食品中的NaNO2。碱性条件下以乙酸锌作为蛋白质沉淀剂纯化提取NaNO2。以C8柱作为分析柱,反相C18柱作为预柱,流动相V(水)∶V(乙腈)=60∶40,荧光检测器在激发波长375 nm发射波长415 nm条件下检测,相关系数r=0.9999,方法检出限为0.01 mg/kg,回收率84.6%~103%,相对标准偏差1.0%~5.0%。方法可用于食品中NaNO2的检测。     

Rapid methods to enumerate Escherichia coli in foods using 4-methylumbelliferyl-beta-D-glucuronide

Three methods to enumerate Escherichia coli in food were compared. They were based on AOAC methods using lauryl tryptose broth (LST) medium, LST-4-methylumbelliferyl-beta-D-glucuronide (MUG) medium, and a proposed method using regular LST in combination with E. coli (EC)-MUG medium. An efficacious and cost-effective method is needed that can detect E. coli and does not produce false presumptive positives. We tested 170 cheeses, 40 frozen processed seafood samples, 210 tree nuts, and 40 other samples. The method of choice for enumerating E. coli depends on the commodity itself. For a product, such as hard cheese or processed seafood, with a history of being negative for E. coli and other lactose-fermenting organisms, the proposed method using regular LST/EC-MUG is a good choice. These samples were seldom presumptive positive in the primary LST medium. If gas was produced, EC-MUG was an effective secondary medium. No false positives (fluorescence) or negatives were detected in EC-MUG medium. For a product with a history of being positive for E. coli and/or other lactose fermenting organisms, such as tree nutmeats or cheeses that are ripened by bacteria or mold, the method using LST-MUG is the method of choice. A presumptive positive in the LST-MUG medium was highly correlative with the biochemical tests that confirmed a sample contain E. coli. For samples spiked with E. coli, the results from each of these 3 methods were identical, and were consistent in enumerating E. coli.     

Chromatographic methods for the determination of pesticides in foods.

Chromatography is the most important technique available to the analyst dealing with the determination of pesticide residues in food, feed and environmental samples. Numerous methods for pesticide residues in foods have been developed in the past few years, and this paper reviews some of the most important procedures. A great variety of chromatographic methods, such as solid-phase extractions, column chromatographic clean-up methods, thin-layer, gas, high-performance liquid and supercritical fluid chromatography, and their coupling with sensitive and selective detection methods are surveyed.     

Precollaborative study of the GeneQuence Salmonella assay using 24-hour enrichment protocols for detection of Salmonella spp. in select foods

New enrichment protocols are described for use with a DNA hybridization (DNAH) method for detection of Salmonella spp. in select foods. GeneQuence Salmonella, in its original version, utilized a 3-stage enrichment of minimum 42 h duration. New 2-stage procedures of 24-28 h duration are described for raw poultry, raw beef, pasteurized egg products, milk chocolate, and dry pet food. In the validation study described here, a total of 345 samples were tested by the abbreviated DNAH method in parallel with either the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA/BAM) or U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) reference culture procedures. Results showed an overall sensitivity for the DNAH method of 97.1% (false-negative rate 2.9%). There were no false-positive results by the DNAH method; therefore the specificity was 100%. Overall agreement between the DNAH and reference culture methods was 98.5%. There were no significant differences in performance between the DNAH and reference methods for any of the foods tested as determined by Chi-square analysis. It is recommended that the DNAH method be subjected to AOAC collaborative study.     

Dry rehydratable film method for rapid enumeration of coliforms in foods (3M Petrifilm Rapid Coliform Count plate): collaborative study

A rehydratable dry-film plating method for coliforms in foods, the 3M Petrifilm Rapid Coliform Count plate method, was compared with the U.S. Food and Drug Administration's Bacteriological Analytical Manual method for nondairy foods and the American Public Health Association's Standard Methods for the Examination of Dairy Products (SMEDP) method for dairy foods. Six food types, vanilla ice cream, cheddar cheese, fresh refrigerated uncooked pasta, wheat flour, prepared frozen macaroni and cheese, and frozen hash browns, were analyzed for coliforms by 11 collaborating laboratories. For each food product tested, the collaborators received 8 blind samples consisting of a control sample and 3 levels of inoculated sample, each in duplicate. The mean log counts for the methods were comparable. The repeatability and reproducibility variances of the Petrifilm Rapid Coliform Count method at 14 and 24 h were not significantly different from those of the standard methods.     

Individual cells immobilization for water-borne pathogen detection and enumeration

A cell counting device has been proposed and implemented for water-borne pathogen detection for drinking water quality monitoring applications. Our approach is based on magnetically-labelled cells immobilization in a high density array of individual cell for optical cell counting. The device has been tested for two water-borne pathogens: Giardia Lamblia & Cryptosporidium. An individual cell immobilization efficiency of 82% was achieved.     

毛细管区带电泳-间接紫外法快速测定食品中的甜蜜素

建立了毛细管区带电泳-间接紫外法快速测定食品中甜蜜素的新方法。液体样品用超纯水稀释后直接进样;固体样品经粉碎或剪碎后用超纯水超声提取后离心,上清液直接进样或用水稀释后进样。以未涂敷石英毛细管（80 cm×75 μm,有效长度：70 cm）为分离柱,以2 mmol/L苯甲酸钠+10 mmol/L碳酸钠+0.5 mmol/L十六烷基三甲基溴化铵为分离缓冲液;于200 nm波长处检测。检出限为8.9 mg/kg （S/N=3）,定量限为26.7 mg/kg （S/N=9）。低、中、高添加水平的加标回收率分别为93.4%、100.3%及101.9%,相应的RSD分别为6.7%、2.0%及2.2%（n=5）。日内及日间精密度分别为2.6%和4.5%。整个分析过程无需有机溶剂。在能力验证样品的分析结果与国家标准方法的结果相吻合的基础上,分析了7件食品样品,获满意结果。     

Comparison of visual immunoassay and chromogenic culture medium for the presence of Listeria spp. in foods

Two rapid screening methods [the TECRA Listeria Visual Immunoassay (LIS-VIS) kit, an AOAC-approved 48 h visual test, which detects Listeria through colorimetry, and BCM Listeria isolation and differentiation plating agar] were used to screen U.S. Food and Drug Administration-regulated commodities for the presence of Listeria spp. Seventy-four different food samples were screened for the presence of Listeria spp. by using both protocols. Test results for the TECRA LIS-VIA showed 66 negative samples and 1 false positive, with 4 confirmed as L. monocytogenes and 3 as L. innocua. With the BCM agar, 67 samples were negative, 4 were confirmed as L. monocytogenes, and 3 were confirmed as L. innocua. Both methods showed similar results and were effective screening tools for Listeria spp. in foods. The BCM agar method proved to be a rapid, sensitive, and excellent tool for early screening and differentiation of Listeria spp. present in foods.     

Validation of three rapid screening methods for detection of verotoxin-producing Escherichia coli in foods: interlaboratory study

An interlaboratory study was conducted for the validation of 3 methods for the detection of all verotoxin-producing Escherichia coli (VTEC) in foods. The methods were a multi-analyte 1-step lateral flow immunoassay (LFIA) for detection of E. coli O157 and verotoxin (VT); an enzyme-linked immunosorbent assay targeted against VT1, VT2, and VT2c (VT-ELISA); and a polymerase chain reaction (PCR) method for detection of VT genes (VT-PCR). Aliquots (25 g or 25 mL) of 4 food types (raw minced [ground] beef, unpasteurized milk, unpasteurized apple juice [cider], and salami) were individually inoculated with low numbers (<9 to 375 cells/25 g) of 6 test strains of E. coli (serogroups O26, O103, O111, O145, and O157) with differing VT-producing capabilities. Five replicates for each test strain and 5 uninoculated samples were prepared for each food type. Fourteen participating laboratories analyzed samples using the LFIA, 9 analyzed the samples by ELISA, and 9 by PCR. The LFIA for O157 and VT had a specificity (correct identification of negative samples) of 92 and 94%, respectively, and a sensitivity (correct identification of positive samples) of 94 and 55%, respectively. The VT-ELISA and VT-PCR had a specificity of 98 and 99%, respectively, and a sensitivity of 89 and 72%, respectively.     

Series analysis methods in enumeration of chemical isomers

Summary Methods of series analysis are used to interpret the results of a computer enumeration of chemical isomers. It is demonstrated how a typical asymptotic behavior that controls the number of isomers arises from singular points of the generating function. The approach is tested on the examples of the isomers of alkanes, alkenes, alkyl radicals and polyenes.     

Petrifilm rapid S. aureus Count Plate method for rapid enumeration of Staphylococcus aureus in selected foods: collaborative study

A rehydratable dry-film plating method for Staphylococcus aureus in foods, the 3M Petrifilm Rapid S. aureus Count Plate method, was compared with AOAC Official Method 975.55 (Staphylococcus aureus in Foods). Nine foods-instant nonfat dried milk, dry seasoned vegetable coating, frozen hash browns, frozen cooked chicken patty, frozen ground raw pork, shredded cheddar cheese, fresh green beans, pasta filled with beef and cheese, and egg custard-were analyzed for S. aureus by 13 collaborating laboratories. For each food tested, the collaborators received 8 blind test samples consisting of a control sample and 3 levels of inoculated test sample, each in duplicate. The mean log counts for the methods were comparable for pasta filled with beef and cheese; frozen hash browns; cooked chicken patty; egg custard; frozen ground raw pork; and instant nonfat dried milk. The repeatability and reproducibility variances of the Petrifilm Rapid S. aureus Count Plate method were similar to those of the standard method.     

Evaluation of the GeneQuence DNA hybridization method in conjunction with 24-hour enrichment protocols for detection of Salmonella spp. in select foods: collaborative study

A multilaboratory study was conducted to compare performance of the GeneQuence DNA hybridization (DNAH) method incorporating new 24 h enrichment protocols and reference culture procedures for detection of Salmonella spp. in select foods. Six food types (raw ground turkey, raw ground beef, dried whole egg, milk chocolate, walnuts, and dry pet food) were tested by the DNAH method and by the culture methods of either the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) or the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA/BAM). Fifteen laboratories participated in the study. Four of the foods tested (raw ground turkey, dried whole egg, milk chocolate, and dry pet food), showed no statistically significant differences in performance between the DNAH method and the reference procedure as determined by Chi square analysis. Sensitivity rates for the DNAH method ranged from 92 to 100%. The DNAH method, with the specific enrichment protocol evaluated, was found to be ineffective for detection of Salmonella spp. in walnuts. For raw ground beef, results from one trial showed a statistically significant difference in performance, with more positives obtained by the reference method. However, evidence suggests that the difference in the number of positives was likely due to lack of homogeneity of the test samples rather than to DNAH method performance.     

Accredited laboratory for detection of irradiated foods in Poland

The history of the development and the role of the Accredited Laboratory for Detection of Irradiated Foods in the national quality food control system are described. The analytical methods for the detection of irradiation in foods adapted and accredited in the Laboratory are enumerated and discussed.     

Rapid and sensitive determination of carbohydrates in foods using high temperature liquid chromatography with evaporative light scattering detection

In the present work, an evaporative light scattering detector was used as a high-temperature liquid chromatography detector for the determination of carbohydrates. The compounds studied were glucose, fructose, galactose, sucrose, maltose, and lactose. The effect of column temperature on the retention times and detectability of these compounds was investigated. Column heating temperatures ranged from 25 to 175°C. The optimum temperature in terms of peak resolution and detectability with pure water as mobile phase and a liquid flow rate of 1 mL/min was 150°C as it allowed the separation of glucose and the three disaccharides here considered in less than 3 min. These conditions were employed for lactose determination in milk samples. Limits of quantification were between 2 and 4.7 mg/L. On the other hand, a temperature gradient was developed for the simultaneous determination of glucose, fructose, and sucrose in orange juices, due to coelution of monosaccharides at temperatures higher than 70°C, being limits of quantifications between 8.5 and 12 mg/L. The proposed hyphenation was successfully applied to different types of milk and different varieties of oranges and mandarins. Recoveries for spiked samples were close to 100% for all the studied analytes.