Detection and Quantification of Enterobacter sakazakii by Real-time 5′-Nuclease Polymerase Chain Reaction Targeting the palE Gene

Enterobacter sakazakii is an emerging food-borne pathogen causing invasive infection with high mortality rates in neonates and infants. The aim of this study was to develop, optimize, and evaluate real-time 5′-nuclease polymerase chain reaction (PCR) for the specific detection and quantification of E. sakazakii. Original primers and TaqMan probe targeting a sequence of E. sakazakii palE gene were designed. The developed real-time PCR system was highly specific for E. sakazakii with 100% inclusivity determined using 54 E. sakazakii strains and 100% exclusivity determined using 99 other strains. Detection limits of 4 × 10² and 4 × 10¹ CFU ml⁻¹ were determined with 100% and 90% probability, respectively. The response of the 5′-nuclease PCR system was linear (correlation coefficient ≥ 0.997) in the range of 10¹ to 10⁸ CFU ml⁻¹. Five methods of DNA sample preparation were compared. The methods of DNA preparation using the InstaGene Matrix and the simple lysis by boiling with the Triton X-100 were the most sensitive with calibration lines applicable for quantification. The developed real-time PCR targeted to the palE gene provides an alternative possibility for the detection and quantification of E. sakazakii after the suitable sample preparation.     

Benzyloxybenzaldehydethiosemicarbazone: Extractive Spectrophotometric Reagent for the Determination of Cu(II) in Food and Water Samples

Benzyloxybenzaldehydethiosemicarbazone (BBTSC) was prepared and developed a new method for the simple, highly selective, and extractive spectrophotometric determination of copper(II) with BBTSC at wavelength 370 nm. The metal ion formed a bluish green colored complex with BBTSC in acetate buffer of pH 5.0, which was easily extractable into n-butanol with 1:1(metal/ligand) composition. The method obeys Beer’s law in the range of 0.5–5.2 ppm. The molar absorptivity and Sandell’s sensitivity were found to be 1.5 × 10⁴ l mol⁻¹ cm⁻¹ and 0.00412 g cm⁻², respectively. The correlation coefficient of the Cu(II)–BBTSC complex was 0.998, which indicated an excellent linearity between the two variables. The repeatability of the method was checked by finding the relative standard deviation (RSD; n = 10), which was 0.377% and its detection limit 0.0204 μg ml⁻¹. The interfering effect of various cations and anions were also studied. The proposed method was successfully applied to the determination of copper(II) in food and water samples. Comparing the results with those obtained using an atomic absorption spectrophotometer tested the validity of the method.     

Improved Sample Preparation for Real-Time PCR Detection of Listeria monocytogenes in Hot-Smoked Salmon using Filtering and Immunomagnetic Separation Techniques

This work evaluated the application of filtration and immunomagnetic separation (IMS) as sample pretreatments for use in combination with real-time polymerase chain reaction (PCR) to detect and quantify Listeria monocytogenes in hot-smoked salmon. Salmon was artificially inoculated with L. monocytogenes at levels ranging from 8 × 10⁰ to 8 × 10⁵ cfu/g of sample, and homogenates obtained from these samples were filtered to recover bacterial cells without a pre-enrichment step. High recovery of bacterial cells was achieved using standard coffee filters. IMS significantly reduced the co-extraction of PCR inhibitors present in the samples to increase the assay sensitivity with regression line parameters applicable for quantification. The limit of detection and quantification were equal to 2 × 10¹–4 × 10¹ and 2 × 10² cfu/g of sample, respectively. The entire detection procedure could be completed within 3.5 h. This study demonstrated that coupling filtration and IMS with real-time PCR has contributed to improve the sensitivity of L. monocytogenes detection from hot-smoked salmon.     

Detection of Salmonella enteritidis and Salmonella typhimurium in foods using a rapid,multiplex real-time recombinase polymerase amplification assay

Salmonella has been recognized as a major foodborne pathogen for humans and animals. In this study, a multiplex real-time recombinase polymerase amplification (RPA) was developed for simultaneous detection of Salmonella enterica serovars, Salmonella enteritidis and Salmonella typhimurium, from chicken, eggs, lettuce, and papaya. The reaction was performed for 20 min at 35°C, and the detection limit of the assay was 10² CFU/ml for pure culture. In food application, the limit of detection (LOD) of S. enteritidis and S. typhimurium using multiplex real-time RPA without enrichment procedure was 10² CFU/25 g, respectively. After enrichment, the LOD of S. enteritidis and S. typhimurium was 10 CFU/25 g. Moreover, the result for Salmonella spp. was not significantly different from those obtained using a culture-based method. Additionally, the assay has a lower cross-reactivity with other pathogenic microorganisms and a good stability performance. Thus, the developed multiplex RPA assay could be used as a rapid tool for the detection of S. enteritidis and S. typhimurium in food.     

Parallel analysis of 7 food-borne pathogens using capillary electrophoresis-based single-strand conformation polymorphism

Capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP) coupled with multiplex polymerase chain reaction (PCR) method was used for the detection of 7 pathogens associated with foodborne illness, including Salmonella enterica, Clostridium perfringens, Bacillus cereus, Listeria monocytogenes, Yersinia enterocolitica, Vibrio parahaemolyticus, and Escherichia coli O157:H7. The method was applied to model food systems, both of culture medium and cooked rice. The detection limit of individual microbes was in the range of 10¹–10³ CFU/g, and that of the mixture of 7 microbes was 10³ CFU/g in the cooked rice sample. This method allowed the detection and identification of all 7 food-borne pathogens within 5 hr without the requirement for enrichment steps.     

Development of a rapid, simple paddle-style dipstick dye immunoassay specific for Listeria monocytogenes

We have developed two types of new paddle-style dipstick dye immunoassays. The first is genus Listeria specific and the second is specific to Listeria monocytogenes. They are based respectively, on antisera raised against heat-killed L. monocytogenes cells and against internalin B crude extract, a virulence protein found only in the pathogenic L. monocytogenes. The minimum detectable level for L. monocytogenes is 2×10⁷ CFU ml⁻¹ for strain number 88/049 in pure culture. Detection is unaffected by the presence of high numbers (approximately log 8.0 CFU/ml) of the other microorganisms tested. When the dipsticks were applied to milk samples inoculated with L. monocytogenes reference material (ALM92), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of milk and ice cream food samples.     

A Sensitive, Selective New Analytical Reagent, 2, 6-Diacetylpyridine bis-4-phenyl-3-thiosemicarbazone for Extractive Spectrophotometric Determination of Mo(VI) in Food Samples

Synthesized a new reagent 2, 6-Diacetylpyridine bis-4-phenyl-3-thiosemicarbazone (2, 6-DAPBPTSC) characterized and is proposed as a sensitive and selective analytical reagent for the extractive spectrophotometric determination of molybdenum(VI) at pH 3.5 to form a yellowish orange colored 1:1 chelate complex. The absorbance was measured at a maximum wavelength, 500 nm. This method obeys Beer’s law in the concentration range 0.90–9.00 μg mL⁻¹ and the correlation coefficient of Mo(VI)–2,6-DAPBPTSC complex is 0.954, which indicates an adequate linearity between the two variables with good molar absorptivity and Sandell’s sensitivity, 1.212 × 10⁴ L mol⁻¹cm⁻¹, 0.0079 μg cm⁻², respectively. The precision and accuracy of the method is checked with calculation of relative standard deviation (n = 5), 0.894% and the detection limit value is 0.0056 μg mL⁻¹. The instability constant of the method has been calculated by Asmus’ method as 6.476 × 10^–5, at room temperature. The interfering effect of various cations and anions has also been studied. The method was successfully applied for the determination of Mo(VI) in food and water samples. The validity of the present method was evaluated in terms of Student ‘t’ test and Variance ‘f’ test, which indicates the significance of the present method an inter comparison of the experimental values, using atomic absorption spectrometer.     

Enzymatic Catalytic Spectrophotometric Determination of Thiamine in Food

A highly sensitive and simple spectrophotometric method for the determination of thiamine based on inhibitory effect of thiamine on the hemoglobin-catalyzed reaction of H₂O₂ with acid chrome blue K was developed. The concentration of thiamine is linear with the percentage inhibition of system under the optimal experimental conditions. The calibration curve is linear in the range 3 × 10⁻⁷ to 3.00 × 10⁻⁵ M with the detection limit of 1.2 × 10⁻⁸ M. This method can be used for the determination of thiamine in food with satisfactory results.     

Determination of Rosmarinic Acid by High-Performance Liquid Chromatography and Its Application to Certain Salvia Species and Rosemary

This study describes a method development for the determination of rosmarinic acid (RA) by using a gradient high-performance liquid chromatography (HPLC) and its application to certain plant materials. The analysis was performed by utilizing a two solvents system [A: methanol/water/formic acid (10:88:2; v:v:v); B: methanol/water/formic acid (90:8:2; v:v:v)] on a reverse-phase column. The flow rate and injection volume were 1 ml min⁻¹ and 10 μl, respectively. Signals were detected at 280 nm. In addition, an internal standard (IS) technique was applied for the analysis of RA to increase precision, and propylparaben was employed for this purpose. The repeatability results as RSD% were 1.66, 1.17 and 1.26 for intra-day and 1.38 was for inter-day with the employment of (3.67 × 10⁻⁵ M) RA. A limit of linearity (LOL) was observed in a wide (1.13 × 10⁻⁵–5.65 × 10⁻⁴ M) concentration range. Linearity parameters were also examined in the range of 5.95 × 10⁻⁶–7.14 × 10⁻⁵ M RA, and very good correlation was observed. The limit of detection (LOD) and limit of quantification (LOQ) (for inter-day) were 1.60 × 10⁻⁶ M (signal/noise [S/N] = 3.3) and 4.80 × 10⁻⁶ M (S/N = 10), respectively. The method was applied to the extracts of certain Lamiaceae plants (Salvia candidissima Vahl. subsp. candidissima, S. sclarea L., S. verticillata L. subsp. verticillata and R. officinalis L.), and reasonable results were obtained.     

Magnetic nano-beads based separation combined with propidium monoazide treatment and multiplex PCR assay for simultaneous detection of viable Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes in food products

We developed a rapid and reliable technique for simultaneous detection of Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes that can be used in food products. Magnetic nano-beads (MNBs) based immunomagnetic separation (IMS) was used to separate the target bacterial cells while multiplex PCR (mPCR) was used to amplify the target genes. To detect only the viable bacteria, propidium monoazide (PMA) was applied to selectively suppress the DNA detection from dead cells. The results showed the detection limit of IMS-PMA-mPCR assay was about 10² CFU/ml (1.2 × 10² CFU/ml for S. Typhimurium, 4.0 × 10² CFU/ml for E. coli O157:H7 and 5.4 × 10² CFU/ml for L. monocytogenes) in pure culture and 10³ CFU/g (5.1 × 10³ CFU/g for S. Typhimurium, 7.5 × 10³ CFU/g for E. coli O157:H7 and 8.4 × 10³ CFU/g for L. monocytogenes) in spiking food products samples (lettuce, tomato and ground beef). This report has demonstrated for the first time, the effective use of rapid and reliable IMS combined with PMA treatment and mPCR assay for simultaneous detection of viable S. Typhimurium, E. coli O157:H7 and L. monocytogenes in spiked food samples. It is anticipated that the present approach will be applicable to simultaneous detection of the three target microorganisms for practical use.     

Loop-Mediated Isothermal Amplification (LAMP) for the Rapid and Sensitive Detection of Salmonella Typhimurium from Pork

ABSTRACT: Loop-mediated isothermal amplification (LAMP) is a novel molecular detection method that is more rapid and simpler than PCR. Products can be detected by turbidity using one temperature without the need for expensive PCR equipment. Our objective was to sensitively detect Salmonella Typhimurium from pork products within 1 d using the LAMP assay. Pork chop and pork sausage samples (25 g) were inoculated with high (10⁸ to 10⁶ CFU) and low (10⁵ to 10⁰ CFU) inocula of S. Typhimurium. Serial dilutions in phosphate buffered saline were plated on XLT4 agar either immediately or after selective preenrichment in tetrathionate broth (225 mL) for 10-h at 37 °C. Nucleic acid was extracted using the TRIzol^® method from 1-mL samples. The LAMP assay using 6 specific invA gene primers and Bst DNA polymerase reaction mix was carried out at 62 °C for 90 min in a waterbath. Turbid products were detected visually and by agarose gel electrophoresis. Improved Salmonella detection at 10² CFU/25 g for both pork chop and sausage was obtained after 10-h enrichment and 10⁶ CFU/25 g without enrichment for both products. This assay can detect Salmonella from pork within 1 d, significantly faster than traditional methods that take >5 d. This method shows tremendous potential for routine diagnostics and monitoring of Salmonella by the pork industry. Practical Application: The novel loop-mediated isothermal amplification (LAMP) assay is a rapid, specific, and sensitive method that has potential application for routine diagnostics of Salmonella from pork products. The isothermal method does not require expensive equipment such as a PCR thermocylcer but only a simple waterbath for amplification within 90 min. Detection is even simpler by visual eye or turbidimeters that are less expensive than fluorescent spectrophotometers or real-time PCR machines. All these advantages make it a practical approach for routine use by processing industries to rapidly detect Salmonella in their environment and to implement appropriate control strategies. To improve detection sensitivities, preenrichment followed by selective enrichment may be necessary. Even so, the entire assay can be completed at the most within two 8-h working shifts.     

A Highly Sensitive Adsorptive Stripping Voltammetric Method for Simultaneous Determination of Lead and Vanadium in Foodstuffs

This work describes a procedure for the simultaneous determination of vanadium and lead in some food and water samples using adsorptive stripping voltammetric method. The method is based on the adsorptive accumulation of cupferron complexes of these elements onto hanging mercury drop electrode, followed by reduction of adsorbed species by voltammetric scan using differential pulse modulation. Optimal analytical conditions were found to be cupferron concentration of 8.00 × 10⁻⁵ M, pH of 4.8 (phosphate buffer), an accumulation potential at −100 mV, and a scan rate of 80 mV s⁻¹. With an accumulation time of 50 s, the peak currents proportional to the concentration of lead and vanadium over the 0.05–80.00 and 0.10–105.00 ng mL⁻¹ ranges with detection limit of 0.02 and 0.01 ng mL⁻¹, respectively. The procedure was applied to simultaneous determination of vanadium and lead in some food and water samples with satisfactory results.     

Spectrophotometric Determination of Trace Nitrite with Brilliant Cresyl Blue Using β-Cyclodextrin as a Sensitizer

A simple and sensitive spectrophotometric method for determination of nitrite has been described. The method is based on the oxidation of brilliant cresyl blue (BCB) by nitrite in acidic medium, which results in the decrease in absorbance at 636 nm. The decrease in absorbance is directly proportional to nitrite concentration obeying Beer’s law. The sensitivity is largely enhanced in the presence of β-cyclodextrin (β-CD) because of inclusion complexation. It was calculated that β-CD and BCB could form 1:1 inclusion complexation with a formation constant of 546.6 L/mol. Linear calibration graphs were obtained for 0.02 × 10⁻³–0.8 × 10⁻³ g/L sodium nitrite at 636 nm. The detection limit for the analytical procedure was 4.0 × 10⁻⁶ g/L sodium nitrite. The relative standard deviation for determination of 0.1 × 10⁻³ g/L and 0.5 × 10⁻³ g/L sodium nitrite were 0.69% and 0.38%, for ten determinations, respectively. Twenty-two coexistent ions or species were examined, and no serious interference for most of ions was observed. The method has been applied to determine nitrite in water and vegetable samples with satisfactory results.     

Improvement of the Detection Sensitivity for <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Spices and Herbs

Various variants of ISO 6888-1:1999 and ISO 6888-3:2003 methods for the detection of Staphylococcus aureus were evaluated and improved for their application to conduct analysis in spices and herbs. Improvement substantiated in washing of the sample to remove compounds interfering with the analysis and in the use of PCR for final detection, instead of plating on Baird-Parker agar, to improve sensitivity at high backgrounds and to save time. The evaluation of the method variants was based on determination of the detection limit (LOD) using series of artificially contaminated spices (allspice, black pepper, cinnamon, nutmeg, paprika, vanilla) and herbs (basil, oregano, parsley, thyme). The method without enrichment, ISO 6888-1:1999, produced LODs of 10³–10⁵ CFU/g with no positive effect of washing the sample or use of PCR for final detection. The method with enrichment, ISO 6888-3:2003, had LOD of 10⁰ CFU/g for basil, black pepper, paprika and parsley. If the washing step was added and PCR was used for final detection, LOD of 10⁰ CFU/g was determined also for cinnamon, nutmeg and vanilla, and LOD of 10¹ CFU/g was determined for allspice. For oregano and thyme, which strongly inhibit the growth of S. aureus, an alternative enrichment-independent method based on direct DNA extraction coupled to real-time PCR may be advantageous.     

Determination of Histamine in Some Foods by Isotachophoretic Method with Simple Sample Preparation

A one-dimensional capillary isotachophoretic method in cationic system of the separation has been applied for histamine determination in food samples. The proposed electrolyte system consisted of 0.01 M potassium hydroxide with l-valine to pH = 9.9 as the leading electrolyte and 0.02 M 2-amino-2-hydroxymethyl-propane-1,3-diol adjusted to pH = 8.3 with 0.1 M hydrochloric acid as terminating electrolyte. Proposed method was characterized by linearity range 5–50 mg L⁻¹ and R ² = 0.9982, accuracy (recoveries ranged from 95% to 102%), detection (2.10 mg L⁻¹), and quantification (7.01 mg L⁻¹) limits. The sample preparation for proposed electrophoretic method included only simple extraction with trichloroacetic acid with filtration and derivatisation stage are avoided. The histamine concentration was determined in meat (turkey, chicken, beef and pork) and meat products (ripened sausage and dry-cured ham), fish (smoked salmon and mackerel), and different kind of mildew and mold ripened cheeses samples. The histamine content ranged from not detected level for fresh meat to 29.63 mg 100 g⁻¹ for cheese samples. The reversed phase HPLC was applied as reference method and the F-Snedecor test and the t test were employed to compare the precision and accuracy of the both methods. Positive correlations were found between the two analytical methods for histamine determination in food products. The obtained results indicate that the proposed electrophoretic method is simple, precise, accurate, and convenient.     

SSEL,a selective enrichment broth for simultaneous growth of Salmonella enterica,Staphylococcus aureus,Escherichia coli O157: H7, and Listeria monocytogenes

Salmonella enterica, Staphylococcus aureus, Escherichia coli O157: H7, and Listeria monocytogenes may contaminate similar types of food and cause foodborne disease. The objective of this study was to develop a selective enrichment broth for simultaneous enrichment of Salmonella enterica, Staphylococcus aureus, Escherichia coli O157: H7, and Listeria monocytogenes (SSEL) using nalidixic acid, acriflavine, lithium chloride, and sodium cholate as selective agents. Developed SSEL broth not only enriched the target pathogens to 5 log10 CFU/ml after 18 hr incubation at 37°C with 10–100 CFU/mL of inoculation concentration, but also could successfully support the simultaneous enrichment of target pathogens with similar growth rates and inhibit the growth of most nontarget bacteria effectively. The enrichment effect of SSEL was confirmed by artificial contamination test coupled with multiplex PCR. In summary, SSEL has been shown to be a promising multiplex selective enrichment broth for the detection of the four pathogens on a single-assay platform.     

Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples

A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of B. cereus CECT 148^T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 3 CFU per reaction or 60 CFU/ml of food, with a relative accuracy of 86.27% to 116.12% in artificially contaminated liquid egg. Naturally contaminated food samples were tested for the presence of B. cereus with the standard method, a conventional PCR and the new developed RTi-PCR assay. Results showed that the new developed RTi-PCR assay is very suitable for detection and quantification of strains of B. cereus group in food samples without an enrichment step.     

Recovery of Native Potato Protein Comparing Expanded Bed Adsorption and Ultrafiltration

Obtaining native protein from potato fruit water (PFW) acceptable for food consumption was attempted by comparing expanded bed adsorption (EBA) and ultrafiltration (UF).The methods were assessed on their process performance and the product quality. Extractable tuber proteins were recovered from lab-prepared PFW either by adsorption to an EBA column using a mixed mode resin (0.31 L) or by batch concentration in an UF (10 kDa MWCO, 0.093 m²) unit and then freeze dried. The yields on protein and esterase activity were higher (p < 0.05 and p < 0.01; Mann–Whitney U-test) in UF (3.2 g l⁻¹ PFW and 3.17 kU l⁻¹ PFW) than in EBA (1.8 and 1.21). The performance difference was also reflected in process productivity for esterase activity which was fivefold better (p < 0.01) in UF (4.30 kU h⁻¹) than with EBA (0.84) due to the higher enzyme retention; protein productivities were the same. The content of solanidine glycoalkaloids was depleted to moderate levels but came out unaffected by the processing method: EBA 286 ppm, UF 213 ppm. The low levels of chlorogenic acid in all EBA preparations were negatively correlated to high brightness score (L* = 73.8%), a favorable attribute in food-quality proteins. Both methodologies produced native preparations of comparable protein content (75%). EBA processing, however, increased the fraction of the patatin protein which may offer advantages in food applications.     

Determination of Melamine in Liquid Milk and Milk Powder by Titania-Based Ligand-Exchange Hydrophilic Interaction Liquid Chromatography

A simple and rapid high-performance liquid chromatography (HPLC) procedure for the analysis of melamine in liquid milk and milk powder has been developed. Decrease of acetonitrile percentage and phosphate buffer concentration in mobile phase, and lowering of buffer pH and column temperature would benefit the retention of melamine on titania. Taking advantage of the ligand-exchange and hydrophilic interaction mixed retention mode on bare titania column, neither complex pretreatment nor ion-pair reagent was required. The whole analysis for one sample including sample pretreatment and HPLC analysis could be accomplished within 30 min. The method presented good linearity (R ² = 0.9998) in a wide range of 0.02–10 μg mL⁻¹. The limit of detection (3σ) and limit of quantification (10σ) of the method were 6 and 20 μg L⁻¹, respectively, which were equivalent to 15 and 50 μg kg⁻¹ melamine in liquid milk, 60 and 200 μg kg⁻¹ melamine in milk powder, respectively. Such sensitivity could be compared with those obtained by HPLC with solid-phase extraction or HPLC coupled with tandem mass spectrometry and was adequate for the screening of melamine in tainted dairy products. The repeatability (RSDs) of the retention times and peak areas of 11 replicate detections of 1.0 μg mL⁻¹ melamine were 0.32% and 2.5%, respectively. The intermediate precision on three consecutive days (RSDs, n = 6) of the retention times and peak areas were 1.1% and 2.3%, respectively. The recovery of spiked melamine in dairy samples ranged from 95.2% to 105%. The simplicity, sensitivity and rapidity of the proposed method make it an effective alternative detecting technique for melamine.