Enterohemorrhagic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EHEC) in water from karst springs: detection with real-time PCR and isolation of strains

Within 2 months, two water sources in a karst area in Switzerland were sampled 9 times each, and analyzed by real-time PCR for 6 EHEC O-types, Shiga-like-toxin (stx1 and stx2) and intimin (eae) genes. With the exception of O111, 5 O-types were recorded regularly and at high frequencies (O26: 33.3 %; O157: 33.3 %; O104: 66.6 %; O103: 72.2 %; O145: 94.4 %). Genes for Shiga-like-toxins and intimin were almost omnipresent (stx1: 77.8 %; stx2: 83.3 %; eae: 77.8 %). Strain isolation was undertaken for O-groups 26, 103, 104, 145 and 157. Sample selection for strain isolation was based on Cq-values for the O-groups and stx1, stx2 and eae. From selected samples, frozen enrichment cultures were cultivated on EHLY-agar and 50 typical colonies screened for the O-type and genes encoding for stx1, stx2 and eae. With this approach, only one virulent EHEC-strain could be isolated (Escherichia coli O103, stx1 +; stx2 ?; eae +). We carried out one extensive testing with 800 colonies of O-group O145, and no virulent strain was isolated. Our findings showed that PCR-results are not sufficient to formulate epidemiological conclusions and that the isolation of strains is necessary. However, as the detection procedure of EHEC in foods is cumbersome and expensive, the appropriateness of such an approach in official food control is a matter of debate.     

Comparison of enrichment procedures for isolation of Escherichia coli O157:H7 from ground beef and radish sprouts

Enrichment procedures using Tryptic soy broth (TSB), modified TSB (mTSB), modified E. coli broth with novobiocin (mEC+n), mTSB (without novobiocin) with vancomycin, cefixime and cefsulodin (mTSB-VCC), or TSB with cefixime, tellurite and vancomycin (TSB-CTV) were evaluated by determining the rate of successful isolation of fifteen Escherichia coli O157:H7 strains from inoculated broth containing ground beef or radish sprout extract. E. coli O157:H7 tended to be isolated more efficiently after enrichment with TSB, mTSB and mEC+n than with the other broths. In order to identify the most efficient enrichmemt condition using these broths, E. coli O157:H7 were inoculated into 25 g ground beef or radish sprouts, which were then homogenized in 225 ml broth and incubated static at 37°C or 42°C for 6 h or 18 h. Attempts were made to isolate the inoculated bacteria by plating method in combination with the immunomagnetic separation method. The most effective enrichment condition was incubation in mTSB or mEC+n at 42°C for 18 h for ground beef, and in mEC+n at 42°C for 18 h for radish sprouts.     

Effect of Radio Frequency Heating on Nalidixic Acid-Adapted Shiga Toxin-Producing and Non-pathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> Strains in Buffer

This study aimed to evaluate the use of nalidixic acid-adapted strains of three major Shiga toxin-producing Escherichia coli (STEC) and non-pathogenic E. coli for the use of radio frequency (RF) heating using phosphate buffer saline (PBS). The effectiveness of RF was evaluated on cocktails of various STEC serotypes (O157:H7, O26:H11, O111) and non-pathogenic E. coli at different endpoint temperatures (55, 60, and 65 °C). All strains were successfully adapted to nalidixic acid (Nal). In general, the results indicated that Nal-adapted strains were not significantly different from Nal-sensitive strains evaluated either before treatment or at the endpoint temperatures. Nal-adapted strains were, therefore, confirmed to be effective as marker organisms in studies involving the use of RF in buffer. Results also showed that the thermal inactivation of strains was more effective as the treatment temperature increased, particularly at 65 °C, which showed a 6.0 log CFU/ml reduction. The results of the present study serve as a baseline to study RF as a potential intervention technology for non-intact beef products.     

Differentiation of big-six non-O157 Shiga-toxin producing Escherichia coli (STEC) on spread plates of mixed cultures using hyperspectral imaging

There have been considerable recent advances in the technology for rapidly detecting foodborne pathogens. However, a traditional culture method is still the “gold standard” for presumptive-positive pathogen screening although it is labor-intensive, ineffective in testing large amount of food samples, and cannot completely prevent unwanted background microflora from growing together with target microorganisms on agar media. We have developed multivariate classification models based on visible and near-infrared hyperspectral imaging for rapid presumptive-positive screening of six representative non-O157 Shiga-toxin producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) on agar plates of pure and mixed cultures. The classification models were developed with spread plates of pure cultures. In this study, we evaluated the performance of the classification models with independent validation samples of mixed cultures that were not used during training and found the best classification model for differentiating non-O157 STEC colonies on spread plates of mixed cultures. A validation protocol appropriate to hyperspectral imaging of mixed cultures was developed. An additional independent validation set of 12 spread plates with pure cultures was used as positive controls to help the validation process with the mixed cultures and to affirm the model performance. One imaging experiment with colonies obtained from two serial dilutions was performed. A total of six agar plates of mixed cultures were prepared, where O45, O111 and O121 serogroups that were relatively easy to differentiate were inoculated into all six plates and then each of O26, O103 and O145 serogroups was added into the mixture of the three common bacterial cultures. The number of mixed colonies grown after 24-h incubation was 331 and the number of pixels associated with the grown colonies was 16,379. The best model found from this validation study was based on pre-processing with standard normal variate and detrending, first derivative, spectral smoothing, and k-nearest neighbor classification (kNN, k = 3) of scores in the principal component subspace spanned by 12 principal components. The results showed 95 % overall detection accuracy at pixel level and 97 % at colony level. The developed model was proven to be still valid even for the independent validation samples although the size of a validation set was small and only one experiment was performed. This study was an important first step in validating and updating multivariate classification models for rapid screening of ground beef samples contaminated by non-O157 STEC pathogens using hyperspectral imaging.     

Bioactive compounds and antioxidant activities of sprout soybean fermented with <Emphasis Type="Italic">Irpex lacteus</Emphasis> mycelia

To enhance the biological activities of sprout soybean, beans were treated with steaming (SS), germinating (GS), or roasting (RS) prior to fermentation with Irpex lacteus mycelia for 20 days. The total phenolic, flavonoid, isoflavone, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of each fermented bean preparation were examined every 5 days for 20 days. The total phenolic content of SS, GS, and RS preparations was 9.61, 10.23, and 10.46 mg/g, respectively, after 15 days of fermentation. These concentrations were approximately 4–5 folds higher compared to initial levels. The total flavonoid content was 8–9 folds higher than initial levels. The isoflavone content was highest in the RS sample (6.84 mg/g). The DPPH radical scavenging activity of beans fermented with I. lacteus mycelia was increased 2–8 folds after 20 days of fermentation. These results indicate that antioxidant activity components were increased by fermentation of I. lacteus mycelia irrespective of soybean treatments.     

Quantification and Discrimination of Viable and Dead <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 Cells from Chicken Without Enrichment by Ethidium Bromide Monoazide Real-time Loop-Mediated Isothermal Amplification

In this study, a rapid and sensitive method of real-time loop-mediated isothermal amplification (Rti-LAMP) assays was developed for quantification and discrimination of viable and heat-killed E. coli O157:H7 cells treated with low concentration of ethidium bromide monoazide (EMA). Four micrograms per milliliter of EMA was chosen as the optimal concentration which did not inhibit DNA amplification derived from viable cells, but significantly increased the Tt values of dead cells in Rti-LAMP assays. When the DNA from 2.0?×?10³ viable CFU of E. coli O157:H7 was subjected to EMA-Rti-LAMP, the resulting Tt value was 17.73 min. In contrast, the DNA from 2.0?×?10³?CFU completely heat destroyed CFU of E. coli O157:H7 did not yield a positive amplification which Tt value was regarded as 60 min. When the DNA from viable plus heat-killed CFU at a ratio of 5:2995 was subjected to EMA-Rti-LAMP, the resulting Tt value was 23.06 min, which was statistically identical (P?<?0.05) to the Tt value of 24.07 min obtained with the DNA from only 5 viable CFU. The results indicate that even though 3.0?×?10³ dead cells yielded a negative amplification setting the Tt value as 60 min, low numbers of viable cells in the presence of much higher numbers of dead cells still yielded a linear plot for enumerating viable CFU from Tt values. Detection of E. coli O157:H7 derived from contaminated chicken samples, the EMA-Rti-LAMP could notably distinguish viable and heat-killed cells from 5.0?×?10¹ to 1.0?×?10⁴?CFU/g without enrichment.     

Occurrence of Shiga toxin-producing E. coli (STEC) on carcasses and retail beef cuts in the marketing chain of beef in Argentina

Argentina has the highest incidence of HUS in the world. HUS is produced by STEC O157 and non-O157. Cattle's faeces and hides are sources of STEC contamination of carcasses during slaughter. We investigated the presence of STEC in carcasses and cuts of meat in the marketing chain in an agricultural city located in Buenos Aires Province (Argentina). In this study, the detection of the stx gene was used as an indicator of carriage of meat with STEC. In carcasses, we detected 12.34% and 18.64% of STEC at the slaughter and sanitary control cabin (place where carcasses arrive from slaughters located outside the city), respectively. These percentages increased at butcheries (24.52%). The 25% of retail beef cuts were STEC-positive with significant differences among the different cuts of meat (chuck: 12.12%, rump roast: 12.12% and minced beef: 40.74%). The stx2 gene was the predominant gene detected in all samples at different levels of the commercialization meat chain.     

Antioxidant Activity and Determination of Phenolic Compounds from <Emphasis Type="Italic">Eugenia involucrata</Emphasis> DC. Fruits by UHPLC-MS/MS

Fruits have been the focus of several studies aimed at finding new antioxidant sources for protection against the damage caused by reactive species. In this study, the antioxidant activity and the presence of phenolic compounds in all parts (peel, pulp, and seeds) of Eugenia involucrata DC. fruits were evaluated. DPPH^·, ABTS^·+, and ORAC methods were used to determine the antioxidant activity, and an UHPLC-MS/MS method was developed for determining the phenolic compounds (gallic, chlorogenic, ferulic, p-coumaric and ellagic acids, quercetin, and myricetin). In the determination of both antioxidant activity and phenolic composition, the efficiency of solvents with different polarities—methanol/H₂O (80:20, v/v), ethanol/H₂O (80:20, v/v), methanol/acidified water with phosphoric acid pH 3.00 (80:20, v/v), and ethyl acetate—for the extraction of the phenolic compounds, was also evaluated. All parts of E. involucrata fruits showed antioxidant activity, in the range of 36.68 ± 1.44 to 873.87 ± 18.24 μmol TE g^?1, being the highest values found in the seeds and peel when more polar extraction solvents were used. Six, five, and three phenolic compounds were identified and quantified in the pulp, peel, and seeds, respectively, with the highest abundance as p-coumaric acid (14 ± 2 mg kg^?1) in the pulp, quercetin (47 ± 5 mg kg^?1) in the peel, and gallic acid (74 ± 4 mg kg^?1) in the seeds, also when more polar solvents were used. Although antioxidant activity methods suggested that the peel and seeds have more antioxidant potential, a wider variety of compounds were determined in the pulp.     

Assessment of Mung Bean Quality Through Single Kernel Characterization

With significant interest in incorporating beans, lentils, and pulses as nutrient-rich healthy food sources into our diets, a reliable technique for their rapid and accurate quality evaluation is needed. The method of single kernel characterization to determine the physical properties (i.e., diameter, weight, moisture content, and hardness) of mung beans was assessed in this study. Two mung bean varieties were characterized using the single kernel characterization technique and the results were compared to traditional methods. It was observed that predicted bean weights were accurate to known laboratory measurements (R = 0.98, n = 200). Individual bean characterization was moderately (R = 0.58–0.7, n = 100) correlated in regard to the true diameter of mung beans. An evaluation on moisture content was performed after tempering the two bean varieties to four moisture levels and a good correlation was obtained with the oven drying method (R = 0.92, n = 24). Hardness values obtained by single kernel characterization were moderately correlated to maximum forces measured using an Instron universal testing system. However, a common relationship was observed between mung bean hardness and moisture content when using both methods. Compared to visual inspection, automated characterization of single beans is a superior technique to measure the geometrical and mechanical properties of mung beans in an industrial setup where high throughput is paramount.     

Rapid Detection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Food Using a Recombinase Polymerase Amplification-Based Assay

Staphylococcus aureus (S. aureus) is of great importance and is a leading cause of food poisoning, which is a public health concern in terms of the frequency and seriousness of the disease. In the present study, RPA and real-time RPA assays were developed and validated to detect S. aureus with high sensitivity and specificity by targeting the nuc gene for the first time. The analytical sensitivity of real-time RPA was 10² copies/reaction, which was higher than the sensitivity of the real-time PCR method. The analysis time was reduced to 10 min, but this method was as reliable as real-time PCR. Furthermore, the potential use of RPA to detect S. aureus was validated with five different artificially contaminated foods. In conclusion, the RPA and real-time RPA assays developed here, similar to real-time PCR, are rapid and simple and exhibit with high sensitivity and specificity. These assays serve as efficient tools for the detection of S. aureus in less advanced laboratories and are suitable for point-of-care detection.     

Effect of Thermosonic Pretreatment and Microwave Vacuum Drying on the Water State and Glass Transition Temperature in <Emphasis Type="Italic">Agaricus bisporus</Emphasis> Slices

This study aimed to understand the micromechanism of thermosonic pretreatment and microwave vacuum drying on Agaricus bisporus. The water state and glass transition temperature (T _g ) of fresh and thermosonically treated Agaricus bisporus slices during microwave vacuum drying were studied using differential scanning calorimetry (DSC), low-field nuclear magnetic resonance (LF-NMR), and magnetic resonance imaging (MRI). Results showed that four population groups were contained in the initial distribution of transverse relaxation time (T ₂) data of fresh A. bisporus slices: T ₂₁ (0.38–7.05 ms), T ₂₂ (9.33–32.75 ms), T ₂₃₁ (37.65–265.61 ms), and T ₂₃₂ (305.39–811.13 ms). Thermosonic pretreatment significantly decreased the initial free water content of A. bisporus sample but was accompanied by a sharp increase in its immobilized water. “Semi-bound water transfer” appeared during microwave vacuum drying (MVD) at moisture contents (X _w ) of 0.70 and 0.60 g/g (wet basis (w.b.)) for untreated and thermosonically treated samples, respectively. MVD caused dramatic changes in the water state and enhanced the T _g by decreasing the content and mobility of immobilized water in A. bisporus tissues. The mobility of semi-bound water for thermosonically and MVD-treated samples was higher than for MVD-untreated samples, resulting in T _g values decreasing by approximately 2–11.5 °C, but the uniformity of water distribution in thermosonic-treated and MVD-treated samples was better at X _w  ≤ 0.52 g/g (w.b.).     

Antibacterial activities of a cinnamon essential oil with cetylpyridinium chloride emulsion against <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium in basil leaves

This study examined the antibacterial activities of two different cinnamon essential oil emulsions against Escherichia coli O157:H7 and Salmonella Typhimurium on basil leaves. Cinnamon oil (0.25%) treatments containing CPC (0.05%) exhibited greater effects on the pathogenic bacteria than cinnamon oil treatment without this emulsifier (p < 0.05). Treatment with cinnamon bark and leaf oil emulsions (CBE and CLE, respectively) reduced the populations of E. coli O157:H7 by 4.10 and 5.10 log CFU/g, and S. Typhimurium by 2.71 and 2.82 log CFU/g, respectively. Scanning electron micrographs showed morphological changes in the two pathogenic bacteria following emulsion treatment. In addition, there was no difference in the color or ascorbic acid content of the basil leaves by the emulsion treatment. These results suggest that CBE or CLE treatment can be an effective way to ensure the microbial safety of minimally processed vegetables and a good alternative to chlorination treatment in the fresh produce industry.     

Detection of Viable <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Cells in Seafood Using a Real-Time Visual Loop-Mediated Isothermal Amplification Combined with Propidium Monoazide

Vibrio cholerae is an important foodborne pathogen causing severe intestinal infectious diseases that have high incidence and mortality. Almost all of rapid testing methods including immunological and molecular assays for V. cholerae are incapable of distinguishing live cells from dead ones, which may overestimate the number of bacteria and result in many false positive results. To address the problems, live cell-specific dye such as propidium monoazide (PMA) is employed. The loop-mediated isothermal amplification (LAMP) assay is a nucleic acid amplification method that is fast, specific, and sensitive. In this study, we developed a real-time visual LAMP assay using PMA dye to detect thyA gene, thereby identifying viable V. cholerae cells. The results showed that only V. cholarae strains could be detected, and there was no cross-reaction with non-V. cholarae strains. Besides, the sensitivity of the PMA-LAMP assay was 1.1 × 10² CFU/mL and the entire reaction could be accomplished within 1 h. The sensitivity was on par with that of the PMA-qPCR assay. The detection limit in different artificially inoculated samples was 5 CFU/25 g materials for the tested pathogens. In the practical test, the PMA-LAMP assay performed well in comparison with PMA-qPCR and the culture method. Hence, PMA-LAMP assay can provide a highly effective and rapid approach for detecting viable V. cholerae.     

Optimization of the QuEChERS Method for Determination of Pesticide Residues in Chicken Liver Samples by Gas Chromatography-Mass Spectrometry

The goal of this research was to evaluate the application of Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method for the determination of organochlorine, organophosphate, and carbamate pesticides in fatty animal matrices such as liver of chicken obtained from National Research Institute of Animal Production in Balice (Poland). Pesticides extraction effectiveness was evaluated at two different spiking levels (0.010 and 0.020 mg kg^?1) and efficiency of the dispersive solid-phase extraction (d-SPE) clean-up step was evaluated by comparison adding different d-SPE sorbent combinations (PSA?+?GCB, PSA?+?C₁₈, PSA?+?SAX, and PSA?+?NH₂). The analysis of pesticide residues was performed by gas chromatography ion trap mass spectrometry (GC/IT-MS). The linear relation was observed from 0 to 400 ng mL^?1 and the determination coefficient R ²?>?0.997 in all instances for all target analytes. Better recoveries were obtained in samples at 0.020 mg kg^?1 spiking level. The recoveries were in the range 70–120 %, with relative standard deviation (RSD) values lower than 15 % at 0.020 mg kg^?1 spiking level for most pesticides. Similar recovery ratios were obtained with the four different combinations of sorbents tested in the clean-up step, with better precision when the (PSA?+?SAX) combination was tested. Limits of detection (LODs) ranged from 0.001 to 0.005 mg kg^?1 and limits of quantification (LOQs) ranged from 0.003 to 0.015 mg kg^?1. The proposed method was successfully applied analyzing pesticide residues in real chicken liver samples; detectable pesticide residues were observed, but in all of the cases, the contamination level was lower than the default maximum residue levels (MRLs) set by European Union (EU), Regulation (EC) N 396/2005.     

Simultaneous Kinetic and Thermodynamic-Based Assay to Determine the Hydrogen Peroxide (H<Subscript>2</Subscript>O<Subscript>2</Subscript>) Scavenging Activity of Berry Extracts by Using Reaction Calorimetry

This work determines the radical scavenging activity of antioxidants and berry extracts based on the heat generated during their reaction with hydrogen peroxide, under isothermal condition (25 °C). After addition of H₂O₂ to a water solution containing antioxidants, an exothermic heat flow appeared. After an initial damping time, the signal decayed exponentially, following a first-order kinetic. Through an iterative fitting routine, both thermodynamic (ΔH) and kinetic (k) information were achieved. Such approach was applied toward relevant food antioxidants, revealing that the fastest reactivity (k) was for tannic acid > gallic acid > caffeic acid > ascorbic acid. Interestingly, k was inversely correlated with ΔH (r = ?0.96) and with the DPPH test (r = ?0.98). Apparently, strong radical scavengers show faster kinetics and lower ΔH-values, as expected, respectively, from a high reactivity toward peroxyl radical and efficient delocalization capacity. Such approach was finally applied to berry extracts (mixed grape seed and skin; chokeberries; grape seed; goji berries). The resulting ΔH-values were correlated with three indices, namely, total phenol, amperometry, and DPPH test. However, k-values largely deviated from these indices. Such discrepancy was explained considering that none of these indices is a “true” measure of the kinetic of the reaction, but only express an apparent concentration. Conversely, reaction calorimetry provides directly and simultaneously both thermodynamic and kinetic properties of the radical scavenging reactivity of antioxidants or natural extracts.     

Effect of Solids Concentration on the Physicochemical and Flow Properties of Cactus Pear Juices of Two Varieties (<Emphasis Type="Italic">Opuntia ficus-indica</Emphasis> and <Emphasis Type="Italic">Opuntia streptacantha</Emphasis>)

Juices from two varieties of cactus pear, a green (Opuntia ficus-indica) and a red (Opuntia streptacantha), were obtained and concentrated by evaporation. Both fruit varieties and their juices at different concentrations were characterized. Green cactus pears had significantly higher amount of pulp than red cactus pears; the peel of O. ficus-indica represented only 38 versus 52 % of the fruit for the O. streptacantha. Both varieties had no significant differences on moisture, density, pH, and titratable acidity, in contrary to soluble solids. Juice was concentrated under vacuum conditions to reach a final concentration of 42, 53–55, and 58–60 °Brix, respectively, and stored under refrigeration (10 °C) during 4 weeks. Physicochemical properties of the pears and juices were determined as fresh items (time zero) and every week for the concentrate juices through storage; similarly, flow parameters were measured at 10 and 25 °C. Concentrate density (1160–1283 kg/m³) was mainly affected by final soluble solids, while pH and acidity were affected differently depending on the variety. Concentrated juices at 42 °Brix were considered with Newtonian behavior with a viscosity of 2–22 mPa s, while those at higher concentrations were of pseudoplastic nature (n < 1.0 and K > 69 mPa sⁿ). Power Law model fitted better the flow behavior than Herschel-Bulkley model of concentrates of both varieties. Temperature, solid concentration, and/or storage time affected the consistency coefficient (K) and flow index (n) depending on the cactus pear variety. Overall, those concentrated juices from O. streptacantha were more stable and exhibited lower apparent viscosity.     

Development and characterization of reconstituted hydrogel from <Emphasis Type="Italic">Aloe vera</Emphasis> (<Emphasis Type="Italic">Aloe barbadensis</Emphasis> Miller) powder

Structural and rheological characterization of reconstituted hydrogels developed from A. vera non-fibrous alcohol insoluble residue (NFAIR) powder using different methods [viz., shaking (S), heating-shaking (HS), and heating (H)] and concentrations (viz., 0.2–1.6 %, w/v) was carried out. Functional group distribution by FTIR spectroscopy and Congo red (CR) method revealed the presence of acetylated acemannan in A. vera powder. Dynamic oscillation studies of A. vera (NFAIR) fluids at all concentrations of 0.2–1.6 %, w/v, showed gel strength in the order of H > HS > S method. However, in H method, increase in concentration from 0.2 to 1.6 %, w/v showed the conformational transition from semi-diluted solution to weak gel nature. Rheological models described the effect of heating temperatures (HT); 30–90 °C, and times (Ht); 15–60 min on viscoelastic behavior in reconstituted A. vera fluids. The reconstituted A. vera hydrogel prepared with a concentration of 1.6 %, w/v using 50 °C (HT) and 30 min (Ht) condition showed a good agreement with the Power law (storage modulus, G′) and Weak gel model (complex modulus, G*) fitted data (R² > 0.94) resulting higher viscoelastic moduli intercepts; G′₀ (71.5 Pa s ^n′), G″₀ (33.5 Pa s ^n″), lower slopes; n′ (0.22), n″ (0.06), higher network strength (A _F , 121.3 Pa s^1/z ) and number of network (z, 5.3) values. The obtained results suggested that heating at 50 °C/30 min can develop aqueous weak gel networks of A. vera with enhanced gel strength which may be utilized as a novel gelling agent for wide variety of targeted applications in food and pharmaceutical sectors.     

Multiplex PCR detection of Shiga toxin-producing Escherichia coli strains belonging to serogroups O157, O103, O91, O113, O145, O111, and O26 experimentally inoculated in beef carcass swabs, beef trim, and ground beef

Numerous foodborne outbreaks are attributed to Shiga toxin-producing Escherichia coli (STEC) and have been recognized for causing gastrointestinal disease in humans. Beef products have been considered the principal source of STEC. A multiplex PCR assay enabling simultaneous detection of STEC O103, O91, O113, O145, O111, O157, and O26 was developed and evaluated in artificially contaminated beef carcass swabs, beef trim, and ground beef after overnight enrichment. Individual serogroups were experimentally inoculated at low (1 to 10 CFU/ml) and high (11 to 100 CFU/ml) levels, and with a cocktail of strains belonging to two, four, and six serogroups. There was no significant difference in detecting single STEC strains under the different conditions. Only when strains were combined were there significant differences in detection of all cocktail isolates in some of the beef products. To address this issue, four serogroups were experimentally inoculated together at three different estimated levels (10, 10(2), and 10(3) CFU/ml) in all three beef products. Results yielded no significant difference in detecting STEC at the three inoculation levels (10, 10(2), and 10(3) CFU/ml) in trim and carcass swabs, but there was a significant difference in detecting STEC at the lowest levels (10 and 10(2) CFU/ml) in the 80:20 nonirradiated ground beef, and in the detection of STEC in irradiated ground beef. The findings from this study could provide industry and government agencies with a tool to evaluate the prevalence and incidence of STEC in beef products and their processing environments.     

Selection of a Taxon-Specific Reference Gene for Qualitative and Quantitative PCR Detection of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis>

Safflower (Carthamus tinctorius) is an emerging model plant for the transgenic modification of fatty acid composition and the production of pharmaceuticals, proteins, or enzymes. Safflower is also a traditional Chinese medicine and is often used as a fake saffron product. Reliable detection of an endogenous reference gene is indispensable for the supervision of genetically modified safflower. Such an endogenous reference gene can also be used to specifically identify safflower ingredient in complex mixtures such as medicine or food. In this study, we identified and validated the CTOS gene as an endogenous reference for safflower. Conventional and real-time polymerase chain reaction (PCR) methods for detecting the CTOS gene sequence showed high interspecies specificity and intra-species stability. The lowest copy number detectable by conventional PCR was 10 haploid copies. The limit of detection and limit of quantification for the real-time PCR assay were estimated to be five and 40 haploid genome copies, respectively. Standard curves established for the real-time PCR assay exhibited good linearity (R ² > 0.99) between the cycle threshold (Ct) values and the initial template copies. The developed conventional and real-time PCR assays were validated in routine analysis of the safflower ingredient in commercial Chinese medicines. In conclusion, the developed quantitative PCR methods were sufficiently specific and sensitive to be used in safflower genomic DNA quantification and safflower ingredient identification.