Detection of Salmonella in Shellfish Using SYBR Green™ I-Based Real-Time Multiplexed PCR Assay Targeting invA and spvB

A SYBR Green? I-based real-time multiplexed PCR assay was developed targeting invA and spvB for the detection of Salmonella strains in shellfish after both hns and invA genes were identified in all Salmonella strains. Simultaneously, the 16S rRNA gene was used as a PCR internal amplification control (IAC). All 89 Salmonella strains tested in this study exhibited amplification of invA, whereas only 21 (23.6 %) were PCR positive for spvB. The sensitivity of detection of all three targeted genes was 1 ng, which is equivalent to approximately 10⁵ colony-forming unit (CFU) of Salmonella enterica. The analysis showed specific PCR products that were identified by reproducible melt temperature profiles (invA, 84.27?±?1.7 °C; spvB, 88.76?±?1.0 °C; and 16S rRNA gene, 87.16?±?0.8 °C).The sensitivity of detection was 10 pg purified DNA (invA) or 10⁵?CFU in 1 mL pure culture of S. enterica ATCC 14028. The above molecular detection method for Salmonella strains was successfully applied to the oyster homogenates (food matrix). An initial inoculum of 10⁶ and 10²?CFU Salmonella in 1 ml seeded oyster tissue homogenate was detected by multiplexed PCR for all three genes after 5 and 24 h of enrichment, respectively. Natural oysters isolated from Gulf of Mexico during the winter months exhibited negative PCR amplification results suggesting the absence of Salmonella. In contrast to conventional PCR, real-time multiplex PCR assay developed in this study is rapid and sensitive and will help Interstate Shellfish Sanitation Conference undertake appropriate measures to monitor Salmonella in oysters, thereby preventing disease outbreaks and consequently protecting consumer health.     

UV–Visible Spectrometry and Multivariate Calibration as a Rapid and Reliable Tool for Simultaneous Quantification of Ternary Mixture of Phenolic Acids in Fruit Juice Samples

Phenolic compounds are biologically important molecules existed in many different plants and fruits. There is a need for a reliable analytical method possessing speedy monitoring, ease of operation, and simple instrumentation. We have developed a fast and reliable spectrophotometric method for simultaneous determination of p-hydroxybenzoic (PHBA), vanillic (VA), and caffeic (CA) acids in fruit juice samples. To overcome the severe spectral overlapping, partial least squares (PLS) regression as a multivariate calibration method was successfully developed for the simultaneous determination of PHBA, VA, and CA in ternary solutions. The experimental calibration matrix was designed with 25 ternary mixtures of these compounds, and the calibration models were validated with six synthetic mixtures. Calibration graphs were linear in the range of 0.0–6.0, 0.0–12.0, and 0.0–12.0 μg mL^?1; limit of detections (LODs) were found to be 0.092, 0.1 l7, and 0.107 μg mL^?1 for PHBA, VA, and CA, respectively. The root-mean-square errors of prediction (RMSEPs) for the same order of target compounds were 0.084, 0.146, and 0.114. The accuracy of the method was confirmed with the recoveries ranging between 84 and 107 %. The relative standard deviations (RSDs) for the analytes in the analysis of fruit juice samples were lower than 4 %. The PLS results were found to be in good agreement with those obtained by high-performance liquid chromatography-ultraviolet detection (HPLC-UV) method. Such a chemometrics-based protocol may be a promising tool for more analytical applications in real sample monitoring, due to its advantages of simplicity, rapidity, and accuracy.     

Taqman PCR-based Methods for Rapid Detection o＇ Salmonella in Pet Food

[Objective] To establish a Taqman real-time PCR for detection of Salmonella in pet food. [Method] A pair of primers and a probe were designed based on published...     

Development an UHPLC-MS/MS Method for Detection of β-Agonist Residues in Milk

β-Agonists have been abused as the growth-promoting agent in food-producing animals over 20 years. The proof of using illegal drugs in food, which is necessary for a regulatory action, usually requires a high degree of specificity and sensitivity. This paper reported an ultra-high performance liquid chromatography (UHPLC)-ESI/MS/MS method for the confirmation of multi-residues of the 13 β-agonist compounds in milk. A wide range of analytes related to β-agonists (brombuterol, cimaterol, clenbuterol, clenpenterol, clorprenaline, hydroxymethylclenbuterol, isoxsuprine, mabuterol, ractopamine, ritodrine, salbutamol, terbutaline, tulobuterol) with similar chemical structures was investigated in order to demonstrate the applicability of our method. This method consists of a two-step extraction and a MCX SPE cleanup. The final extract was separated by UHPLC within 5 min and then injected in an electrospray ionization mass spectrometry for the determination. Using clenbuterol-D9, salbutamol-D3, and ractopamine-D5 as internal standards, and accomplishing with the matrix matched calibration curves to compensate for the matrix effects, the quantitative data showed good linear response within the concentration ranges studied. The detection limits (CCα) and detection capabilities (CCβ) of the analytes were found in the range of 0.01–0.16 μg/L and 0.03–0.21 μg/L, respectively. Recoveries of the compounds were found from 82.5% to 101% at the spiked level of 0.05–2.5 μg/L, and the relative standard deviation was within the range of 7.17% and 16.4%. Furthermore, an inter-laboratory study among eight laboratories was conducted to further validate the method, and the results were found satisfactory.     

A Robust and Poisson Validated Quantitative 5′ Nuclease TaqMan® Real-Time PCR Assay Targeting fimA for the Rapid Detection of Salmonella spp. in Food

A newly designed TaqMan® probe and an internal amplification control were implemented in a conventional PCR system targeting the major fimbrial subunit encoding gene fimA. This assay has an inclusivity and exclusivity of 100 % (n?=?126). The limit of detection (LOD) and the absolute quantification limit, both determined by advanced Poisson analyses, were three bacterial cell equivalents per quantitative real-time PCR reaction. The fimA assay achieved 100 % accuracy and performed very robustly, producing reproducible, reliable data for quantification of Salmonella spp., even at the LOD.     

Relative Accuracy,Specificity and Sensitivity of a 5′ Nuclease Real-Time PCR Assay for Listeria monocytogenes Detection in Naturally Contaminated Pork Cuts

In the present study, the relative sensitivity, specificity and accuracy of a real-time PCR assay for Listeria monocytogenes detection in naturally contaminated pork cuts were evaluated in comparison to the ISO 11290-1:2004 reference culture method. One hundred and sixty meat samples were collected from ten different lots over a year. The PCR method included a 24-h primary enrichment step, a DNA extraction step, and a final 5′ nuclease real-time PCR assay, including an internal amplification control (IAC) and targeting the hlyA gene. Based on the analysis of 480 sub-units (three sub-units for each sample), the relative sensitivity, specificity and accuracy of the real-time PCR assay were 77.3 %, 67.0 % and 73.1 %, respectively, corresponding to a Cohen’s kappa value of 0.69 (good agreement). Considering that real samples from the worse storage scenarios were included, these results suggest the PCR method as a rapid and accurate alternative method for the quick check of L. monocytogenes in meat lots before distribution.     

High pressure inactivation of Salmonella on Jalapeño and Serrano peppers destined for direct consumption or as ingredients in Mexican salsa and guacamole

In summer of 2008, the United States witnessed one of the largest multi-state salmonellosis outbreak linked to the consumption of Jalape?o and Serrano peppers tainted with Salmonella enterica serovar Saintpaul. The first objective of this study was to assess the application of high hydrostatic pressure (HHP) to decontaminate Jalape?o and Serrano peppers from this pathogen. Jalape?o and Serrano peppers were inoculated with a five-strain cocktail of Salmonella to a final level of ca. ~6 log CFU/g and subsequently pressure-treated in the un-wetted, wetted (briefly dipped in water) or soaked (immersed in water for 30 min) state at 300-500 MPa for 2 min at 20°C. The extent of pressure inactivation increased as a function of the pressure level and in the order of soaked>wetted>un-wetted state achieving population reductions ranging from 1.1 to 6.6 log CFU/g. Overall, pressure treatment at 400-450 MPa (soaked) or 450-500 MPa (wetted) for 2 min at 20°C rendered Salmonella undetectable. Since salsa and guacamole are two examples of widely consumed Mexican dishes that incorporate raw Jalape?o and Serrano peppers, we subsequently investigated the pressure-inactivation of Salmonella in salsa and guacamole, originating from contaminated peppers used as ingredients. The storage time (0, 12 or 24 h) of the condiments prior to HHP as well as the pH (3.8-5.3) and the type of acidulants (vinegar and lemon juice) used all influenced the extent of Salmonella inactivation by HHP. This study demonstrates the dual efficacy of HHP to decontaminate fresh chile peppers destined for direct consumption and minimally process condiments possibly contaminated with raw peppers to enhance their microbiological safety.     

Validation of a LC–MS Method for the Determination of Urea Contamination in Market Teas

Tea is one of the most widely consumed beverages in the world preceded only by water. In some culture style of tea plant, urea fertilizer is sprayed on tea leaf in order to increase crop output; besides, in some processing method of green tea, urea is illegally added to maintain tea’s vivid color. The above-mentioned process may unavoidably cause urea residue, and prolonged or repeated exposure of body to urea may cause adverse effects. In this work, an easy and reliable hydrophilic interaction liquid chromatography/mass spectrometry analytical method for determination of urea in tea was firstly validated; the method exhibited a linear response from 4 to 60 μg mL^?1 (R ²?>?0.9996), the limit of quantitation for urea is 4 μg mL^?1, the mean recoveries of urea spiked at levels of 10 and 20 μg mL^?1 were 95–110 %, and the relative standard deviations of intra- and inter-day measurements were less than 7.8 %. The method was later successfully applied to the analysis of urea residue in some market tea samples. The result showed that there is obvious urea contamination in some market teas, and the contamination percentage is especially high in green tea samples. Among the samples investigated, urea was detected at concentration ranges of 24.02–46.02 mg/kg. Some attention should be paid on the health effect resulting from such urea contamination in tea.     

Validation of a Method for Analysis of Aroma Compounds in Red Wine using Liquid–Liquid Extraction and GC–MS

An analytical method for determination of volatile composition of wines using sample preparation by liquid–liquid extraction and gas chromatography coupled to mass spectrometry for separation and detection has been developed and validated. Extraction of volatile compounds was performed in dichloromethane, and 1-octanol was added as an internal standard. Kékfrankos red wine produced in Villány wine region in Hungary was used as a model wine for testing and validation of the method. The developed method allowed satisfactory determination of 33 volatile compounds in the wines. Compounds analyzed include alcohols, esters, lactones, fatty acids, furans, and nitrogen compounds. The calibration curves of the four reference compounds used (2-phenyl ethanol, ethyl nonanoate, butyrolactone, and tyrosol) were linear in all cases with correlation coefficients (R ²) ranging from 0.9951 to 0.9992. The accuracy of the method was checked with a standard addition method (recovery 92.2–103 %), showing good repeatability and reproducibility (RSD?<?10 %).     

Design of a combined process for the inactivation of Salmonella Enteritidis in liquid whole egg at 55°C

This paper is an evaluation of the lethal effectiveness of a successive application of pulsed electric fields (PEFs) and heat treatment in liquid whole egg (LWE) in the presence of different additives on the population of Salmonella serovar Enteritidis. Synergistic reductions of the Salmonella Enteritidis population were observed when LWE samples containing additives were treated with PEF (25 kV/cm; 100 and 200 kJ/kg), heat (55 °C), or PEF followed by heat. The presence of additives, such as 10 mM EDTA or 2% triethyl citrate, increased the PEF lethality 1 log?? cycle and generated around 1.5 log?? cycles of cell damage, resulting in the reduction of undamaged cells of 4.4 and 3.1 log?? cycles, respectively. The application of PEF followed by heat treatment significantly (p < 0.05) reduced D(55 oC) from 3.9 ± 0.2 min in LWE to 1.40 ± 0.06 min or 0.24 ± 0.02 min in the presence of 10 mM EDTA or 2% triethyl citrate, respectively. A PEF treatment of 25 kV/cm and 200 kJ/kg followed by a heat treatment of 55 °C and 2 min reduced more than 8 log?? cycles of the population of Salmonella Enteritidis in LWE combined with 2% triethyl citrate, with a minimal impact on its protein soluble content. The heat sensitizing effect of PEF treatments in the presence of 2% triethyl citrate on the Salmonella population could enable LWE producers to reduce the temperature or processing time of thermal treatments (current standards are 60 °C for 3.5 min in the United States), increasing the level of Salmonella inactivation and retaining the quality of non-treated LWE.     

Survival of Salmonella Typhi and Shigella dysenteriae in dehydrated infant formula

Abstract: Powdered infant formula has previously been linked to the transmission of various bacterial pathogens in infants resulting in life‐threatening disease and death. Survival studies of 2 common foodborne pathogens, Salmonella enterica serovar Typhi and Shigella dysenteriae, in powdered infant formula have not been previously studied despite the potentially devastating consequences from ingestion of these organisms, particularly by newborns, in case of a natural or deliberate contamination event. Therefore, to better predict the risk of S. Typhi and S. dysenteriae infection from consumption of infant formula, the present study was undertaken to determine survival of these microorganisms in dry infant formula under varying atmospheric conditions. A 2‐strain cocktail of S. Typhi and a 3‐strain cocktail of S. dysenteriae were stored for up to 12 wk in dehydrated infant formula in an ambient air or nitrogen atmosphere. Viable counts of S. Typhi at 12 wk in infant formula revealed a 2.9‐ and 1.69‐log decrease in ambient air and nitrogen atmosphere, respectively. Viable counts of S. dysenteriae at 12 wk in infant formula revealed a 0.81‐ and 0.42‐log decrease in ambient air and nitrogen atmosphere, respectively. These results show that S. Typhi and S. dysenteriae can remain viable for prolonged periods of time in powdered infant formula, and the presence of nitrogen enhances survival. Practical Application: Our goal in this work was to study the survival of S. Typhi and S. dysenteriae in dehydrated storage conditions in infant formula. This interest is partially generated by the possibility of using these 2 microorganisms to deliberately contaminate the food supply. The outcome of this study will help us to have a better idea how to respond and react to the risk of deliberate food contamination.     

Mass Fragmentation Studies of α-Tomatine and Validation of a Liquid Chromatography LTQ Orbitrap Mass Spectrometry Method for Its Quantification in Tomatoes

Tomatoes, members of the Solanaceae plant family, produce biologically active secondary metabolites, including glycoalkaloids, which may have both adverse and beneficial biological effects. By using the linear ion trap (LIT) mass spectrometry, multistage collision-induced dissociation (CID) experiments (MS ⁿ ) were performed to elucidate characteristic fragmentation pathways of the glycoalkaloid α-tomatine. High-resolution with high-accuracy mass analysis using an Orbitrap Fourier transform MS (FTMS) with higher-energy CID (HCD) was used to produce mass spectra data across a wide spectral range for confirmation of proposed ion structures and formulae. In addition, a new liquid chromatography method that utilized LTQ Orbitrap MS was developed for the analysis of α-tomatine in tomatoes. Recoveries of α-tomatine were >96.0 % with relative standard deviations (RSD) below 7.98 %. The limit of detection (LOD) was 0.002 mg/kg. The limit of quantitation (LOQ) was 0.005 mg/kg. The linear range was between 0.010 and 10 mg/kg with an excellent correlation coefficient (R ²)?≥?0.9991. Various tomato samples were analyzed for method application, and the level of α-tomatine in the 11 samples analyzed ranged from 0.0011to 0.3077 mg/kg.     

Validation of an Electrochemical Detection–High-Performance Liquid Chromatography Method for Simultaneous Determination of Lignans in Flaxseed (Linum usitatissimum L.)

Flaxseed is a major source of lignans, which are important bioactive compounds. The aims of this work were to validate a liquid chromatographic method for the rapid and simultaneous determination of the main lignans in flaxseed using high-performance liquid chromatography coupled with electrochemical detection and to analyze the composition of commercial samples of flaxseed. The performance criteria of the proposed method demonstrate that the method can be used for the analysis of secoisolariciresinol diglucoside (SDG), secoisolariciresinol (SECO), matairesinol (MATA), pinoresinol (PINO), lariciresinol (LARI), hydroxymatairesinol (HYDROXY), and isolariciresinol (ISOLARI) in flaxseeds at suitable levels. Calibration curves were determined for six different concentrations of standard solutions injected in triplicate. The sensitivity of the calibration curve was evaluated considering the confidence intervals of the intercept and slope. The limit of detection and limit of quantification were 7.4 and 10.9 μg/l, respectively, for LARI and 17.7 and 37.5 μg/l, respectively, for MATA. The relative standard deviation of repeatability values were lower than 2.59 %, which are acceptable because the Horwitz ratio values were 0.1 for all of the lignans. The recoveries of lignans were in the range of 74–100 % of SECO, which are consistent with the literature. The precision of the proposed method was determined by analyzing four flaxseed samples of different years and varieties. SDG was the main lignan present in all the samples, followed by ISOLARI and HYDROXY.     

Salmonella surveillance and control for finisher pigs and pork in Denmark — A case study

Salmonella can either be controlled pre-harvest, post-harvest or by a combination of both approaches. This paper describes the lessons learned in Danish Salmonella surveillance and control programme for finisher pigs and pork. Initially, main focus was on pre-harvest initiatives and correct identification of herds with respect to the risk for Salmonella that they represented. However, an analysis of risk-mitigating actions applied along the chain from stable to table showed that it would be more cost-effective to deal with Salmonella on the abattoirs than in the herds. This knowledge moved focus from pre- to post-harvest without giving up on pre-harvest surveillance. First of all, this meant increased attention on slaughter hygiene and individual interventions in the abattoirs. In brief, we learned that for a programme to be successful it must be based on standardised methods for sampling and testing to be able to evaluate and compare performance of the programme. More specifically, meat-juice samples taken from finisher pigs at the time of slaughter are an effective way of identifying high-risk herds for Salmonella. In addition, a penalty system might act as an incentive for farmers to deal with Salmonella in their herd. Additionally, common targets for all abattoirs allowing for unique control solutions should be adapted. Finally, decontamination techniques like hot water decontamination are a feasible way of dealing with high-risk pigs (Level-3 pigs). The current prevalence in Danish pork is around 1.2%, and a target is set to < 1.0% to be reached by the end of 2013. The experience obtained by use of the Danish programme might be used to develop and implement appropriate types of surveillance programs as well as risk-mitigating measures in other countries.     

Development and validation of a predictive microbiology model for survival and growth of Salmonella on chicken stored at 4 to 12 °C

Salmonella spp. are a leading cause of foodborne illness. Mathematical models that predict Salmonella survival and growth on food from a low initial dose, in response to storage and handling conditions, are valuable tools for helping assess and manage this public health risk. The objective of this study was to develop and to validate the first predictive microbiology model for survival and growth of a low initial dose of Salmonella on chicken during refrigerated storage. Chicken skin was inoculated with a low initial dose (0.9 log) of a multiple antibiotic-resistant strain of Salmonella Typhimurium DT104 (ATCC 700408) and then stored at 4 to 12 °C for 0 to 10 days. A general regression neural network (GRNN) model that predicted log change of Salmonella Typhimurium DT104 as a function of time and temperature was developed. Percentage of residuals in an acceptable prediction zone, from -1 (fail-safe) to 0.5 (fail-dangerous) log, was used to validate the GRNN model by using a criterion of 70% acceptable predictions. Survival but not growth of Salmonella Typhimurium DT104 was observed at 4 to 8 °C. Maximum growth of Salmonella Typhimurium DT104 during 10 days of storage was 0.7 log at 9 °C, 1.1 log at 10 °C, 1.8 log at 11 °C, and 2.9 log at 12 °C. Performance of the GRNN model for predicting dependent data (n=163) was 85% acceptable predictions, for predicting independent data for interpolation (n=77) was 84% acceptable predictions, and for predicting independent data for extrapolation (n=70) to Salmonella Kentucky was 87% acceptable predictions. Thus, the GRNN model provided valid predictions for survival and growth of Salmonella on chicken during refrigerated storage, and therefore the model can be used with confidence to help assess and manage this public health risk.     

Capillary Isoelectric Focusing—Useful Tool for Detection and Quantification of Lactic Acid Bacteria in Milk

The lactic acid bacteria (LAB), including lactobacilli and enterococci, represent an important part of normal microflora in humans. Simultaneously, they are frequently used as probiotics and in food industry for production of fermented milk products. The rapid and sensitive detection of these microorganisms is crucial for the quality control. In this study, the capillary isoelectric focusing (CIEF) was successfully used for the separation and characterization of probiotic LAB, L. paracasei sp. paracasei; L. acidifarinae; L. fermentum; L. gasseri; L. helveticus; L. plantarum; L. delbrueckii; L. salivarius; E. durans; E. faecalis; and E. faecium, according to their isoelectric points (pI). All pIs of lactobacilli and enterococci were found in the acidic part of the pH range. Subsequently, the milk samples spiked with L. rhamnosus, and E. faecium were analyzed by CIEF. The optimal adjustment of the separation conditions allowed detection and quantification of the bacteria in the sample with sufficient sensitivity. Therefore, CIEF is an efficient approach to rapid detection and separation of LAB even directly in milk products in future.     

Detection and identification of wild yeasts in Champús, a fermented Colombian maize beverage

The aim of this study was to identify and characterise the predominant yeasts in Champús, a traditional Colombian cereal-based beverage with a low alcoholic content.Samples of Champús from 20 production sites in the Cauca Valley region were analysed. A total of 235 yeast isolates were identified by conventional microbiological analyses and by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of ITS1-5.8S rDNA-ITS2. The dominant species were: Saccharomyces cerevisiae, Issatchenkia orientalis, Pichia fermentans, Pichia kluyveri var. kluyveri, Zygosaccharomyces fermentati, Torulospora delbruekii, Galactomyces geotrichum and Hanseniaspora spp. Model Champús systems were inoculated with single strains of some isolated sporogenus species and the aromatic profiles were analysed by SPME. Analysis of data showed that Champús strains produced high amounts of esters. The aromatic compounds produced by Saccharomyces and non-Saccharomyces yeasts from Champús can exert a relevant influence on the sensory characteristics of the fermented beverage. The Champús strains could thus represent an important source for new yeast biotypes with potential industrial applications.