Spiked Food Samples Research Articles

Protein and DNA-based assays as complementary tools for fish allergen detection.

Background: Fish is one of the most important, allergenic foods worldwide. Parvalbumin is the well characterized, major allergen in fish muscle. In this study, we developed a protein- and a DNA-based method for the sensitive detection and authentication of eight commonly consumed fishes in food and compared their applicability. Methods: Fish parvalbumins were purified. Polyclonal, anti-parvalbumin antibodies were raised in rabbits and mice. Protein extracts from food were analyzed by quantitative ELISA. Parvalbumin genes were cloned and sequenced for the design of parvalbumin gene-specific PCR-primers. DNA extracted from food was subjected to specific PCR. Results: Increasing parvalbumin contents were quantified by ELISA in fresh fish, in the order of tuna < mackerel < cod < salmon/trout < redfish < carp < herring. The parvalbumin content of processed fish was up to 67% lower than in fresh fish. In spiked food samples, 1 to 15 ppm fresh fish and 30 to 170 ppm processed fish were still detectable by ELISA. The eight fishes were identified by specific PCR using 0.2 to 10 ng fish DNA. PCRs detected still 3 ppm fresh fish and 30 to 150 ppm processed fish in spiked samples. Conclusions: Both the protein- and the DNA-based method have sufficient sensitivity to protect fish-allergic consumers. The ELISA allows allergen quantification, while the PCR identifies the fish present in the food. The detection limits of both methods vary depending on different factors. Both methods need to be carefully validated for each fish and fish product when used in detection assays.

Performance Evaluation and Thermodynamic Studies for the Simultaneous Cloud Point Extraction of Erythrosine and Tartrazine Using Mixed Micelles in Food Samples

The present method describes simultaneous determination of erythrosine and tartrazine after cloud point extraction using mixed micelles. The coacervate phase containing the analytes was determined spectrophotometrically by measuring the absorbance at 525 and 425 nm for erythrosine and tartrazine, respectively. Different parameters such as pH, concentration of nonionic (Triton X-100) and cationic surfactant (cetyl trimethyl ammonium bromide (CTAB)), electrolyte concentration, temperature, and incubation time were suitably optimized. Among the various parameters studied, pH and the nature of surfactants played a major role in efficient cloud point extraction of the analytes. A mixture of Triton X-100 and CTAB provided adequate separation efficiency, with enrichment factor of 45.22 and 71.15 for erythrosine and tartrazine, respectively. The method was found to be linear in the concentration range of 0.04–4.00 μg mL−1 for erythrosine and 0.05–5.00 μg mL−1 for tartrazine. The limit of detection and limit of quantitation obtained were 11.24 and 37.19 μg L−1 for erythrosine and 13.33 and 44.27 μg L−1 for tartrazine, respectively. The recovery of these dyes from spiked food samples ranged from 96.5–103.9%. Considering a homogenous monolayer adsorption in the phase separation, Langmuir isotherm model was applied. Thermodynamic parameters such as free energy, enthalpy, and entropy were determined from the plot of solubilization affinity against the reciprocal of temperature. The effect of cations, anions, salts, and some food colorants on the determination of erythrosine and tartrazine was also studied. To demonstrate method applicability, erythrosine and tartrazine were determined in fruit jelly, fruit candy, and fruit syrup samples.

Competitive photometric and visual ELISA for aflatoxin B1 based on the inhibition of the oxidation of ABTS

This work reports on a sensitive colorimetric immunoassay for aflatoxin B1 (AFB1) in foodstuff. The reagent L-ascorbic acid 2-phosphate (AAP) is added to the system, and alkaline phosphatase (ALP) hydrolyzes AAP under formation of ascorbic acid and phosphate. The ascorbic acid produced reduces chloroauric acid to form zero-valent gold (in the form of nanoparticles). Hence, Au(III) is no longer available to oxidize 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) to form a green product. Rather, the solution remains colorless. By using ALP-labelled monoclonal anti-AFB1 antibody, a competitive enzyme-label immunoassay was developed for AFB1 in a microplate coated with the AFB1-BSA conjugate. Under optimal conditions, the absorbance of the solution at 415 nm increases linearly with increasing AFB1 concentration in range from 10 pg·mL−1 to 100 ng·mL−1 (while the color gradually turns to green), and the detection limit is 7.8 pg·mL−1. The precision of the method (expressed as RSD) is ±9.7%. The accuracy was validated by analyzing both naturally contaminated and spiked food samples, and the results matched the results obtained by ELISA very well.

Developing fast laboratory screening platform for sulfate dioxide in food by surface-enhanced Raman spectroscopy

The direct detection of sulphur dioxide has been realized in various food matrixes with high sensitivity and high selectivity, on the basis of the integration of the high energy resolution of the finger-print spectrum of surface-enhanced Raman spectroscopy and the universal but easy-on-going pretreatment procedure. The characteristic peak of sulphur dioxide at ~630 cm−1 was applied as the qualitative and quantitative standard, which displayed a lowest detectable concentration at the 1 mg kg−1 level for the spiked food samples. The key point of the high sensitivity and selectivity is the effective pretreatment born out of the standard distillation one, which has been improved in the three parts. (1) Using CaCO3 and Zeolite instead of N2 gas as the bubbling reagent. (2) Using oxalic acid as the acidic distillation solution to eliminate the hazards from acid volatilization, such as hydrochloric acid. (3) Using diluted sodium hydroxide solution as the absorption reagent instead of lead acetate solution to avoid the secondary pollution. With the three distinguished advantages of environment friendly, high sensitivity and free of matrix interference, the proposed method has great potential to replace the traditional ones for the fast screening of the illegal or abused sulphur dioxide in food.

Development and validation of a SYBR-Green I Real-Time PCR test to detect bivalves includingMytilusspecies in foods

Summary The incidence of allergy to seafood, and in particular to molluscs is second only to that of nuts. To protect consumers, the regulators of food products insisted on identifying molluscs as allergens. The aim was to develop quantitative assay for the presence Mytilus species in processed food products. The chosen platform was real-time PCR (qPCR) targeting either the gene encoding mitochondrial cytochrome C oxidase I or the nuclear gene encoding β-actin. Recombinant plasmids containing each of target regions were used as a reference for quantification purposes. Limit of detection (LOD) and of quantification (LOQ) were determined. Spiked food samples containing 50–500 μg g−1 of Mytilus chilensis were analysed both by qPCR and by ELISA. The former assay gave a positive outcome over this range, whereas the latter was sensitive down to a concentration of 125 μg g−1.

Development of a Polymerase Chain Reaction-Temporal Temperature Gradient Gel Electrophoresis Assay for Identification of Salmonella enterica Subspecies enterica Using a Hypothetical Non-specific Endonucleas S. entericae Gene Sequence

Background: Salmonella is one of the major agents of food-borne diseases that cause severe illness in humans. The conventional detection methods of these bacteria are time-consuming with low sensitivity and specificity, which limit their applications. Therefore, developing more rapid and accurate methods is urgently required in food safety programs. Objectives: In this study for the first time, polymerase chain reaction-Temporal temperature gradient gel electrophoresis (TTGE) was optimized for the identification of Salmonella enterica subspecies enterica serovars in processed food samples using a single-copy sequence. Methods: DNA was isolated from pure cultures of Salmonella and non-Salmonella strains. The single copy target sequence was selected and amplified by employing the polymerase chain reaction (PCR) with specific primers, designed in this study, and their specificity and sensitivity were determined. The TTGE parameters, especially the temperature gradient and the time of reaction, were optimized and then this method was applied to investigate spiked food samples. Results: The PCR detection sensitivity was recorded as 12 × 103 CFU/mL in artificially-contaminated food samples. The best resolution was observed at a temperature gradient from 62.5 to 67.5°C, with a ramp rate of 1°C/hour and electrophoresis for 5 hours at a constant voltage of 130 V. The TTGE patterns obtained from the artificially-contaminated samples were similar with the respected standard bacteria strains. The optimized TTGE protocol resulted in the separation of the same length PCR products into different band positions on the polyacrylamide gel. Conclusions: By optimization of PCR-TTGE and determination of distinct band pattern for standard bacteria, this method can be used as a fast screening test to investigate the presence of S. enterica, subspecies enterica, directly in food samples.

In vitro selection of DNA aptamers targeting β-lactoglobulin and their integration in graphene-based biosensor for the detection of milk allergen

Food allergy has increased rapidly in recent years affecting millions of people worldwide. With the increased consumption of packed food nowadays, a sensitive, accurate and rapid screening method for potential food allergens has become an urgent need in order to protect the sensitive consumers from life-threatening reactions. The current detection methods for food allergens are mostly based on immunoassays which are costly and times-consuming. In this work, we developed an aptamer/graphene-based electrochemical biosensor for on-step, sensitive and low cost detection of β-lactoglobulin (β-LG) milk protein, one of the most common food allergens specially in infants. The selection of DNA aptamers against the two β-LG variants A and B was successfully realised using systemic evolution of ligands by exponential enrichment (SELEX) method. Among the selected aptamers, BLG14 aptamer sequence has shown high affinity and specificity to both β-LG A and B with dissociation constants (Kds) of 82 and 80nM, respectively as calculated using fluorescence binding assays. The aptamer was then integrated in a voltammetric biosensor utilizing graphene-modified screen printed carbon electrodes. The binding is monitored by following the change in the square wave voltammetry (SWV) reduction peak signal of ferrocyanide/ferricyanide redox couple due to the removal of the negatively charged aptamers from the surface upon protein binding. This one-step "signal on" biosensor takes 20min for the sensitive and selective detection of β-LG without using any labelling or sophisticated designs. The method was also tested in spiked food sample extract achieving good recovery percentage. The integration of the novel aptamer in the graphene biosensor allows a promising way for cost-effective, rapid and sensitive on-site detection of milk allergen in food stuff.

Development of a multiplex real-time recombinase polymerase amplification (RPA) assay for rapid quantitative detection of Campylobacter coli and jejuni from eggs and chicken products

Chicken products are known as main vehicles for human infections and foodborne illness with campylobacteriosis. This study describes a rapid and sensitive method for the detection of C. coli and C. jejuni from eggs, chicken meat, and chicken broth using the multiplex real-time recombinase polymerase amplification (Rti-RPA) and its comparison to a culture dependent method. The optimal procedure for the multiplex Rti-RPA assay was set in isothermal condition at 45 °C for 20 min and the detection sensitivity was at 1 CFU/reaction in pure culture. In the case of food applications, the detection limit of C. coli and C. jejuni using the RPA assay was 1 CFU/mL from chicken broth, 103 CFU/g from egg and chicken meat samples without a pre-enrichment procedure, and 1 CFU/g from 24 h enriched egg and chicken meat samples. Quantifications of Campylobacter spp. in spiked food samples by the RPA assay were not significantly different with the results by the culture method. This multiplex Rti-RPA assay is considerably faster than other DNA amplification methods and exhibits no losses in detection sensitivity and specificity. This multiplex Rti-RPA is a simple, rapid detection and quantification assay that is completed within a 20 min runtime. The assay is applicable in the surveillance of C. coli and C. jejuni contamination in eggs and chicken products.

Application of Time-resolved Fluroimmunoassay for Determination of Furaltadone Metabolite 3-Amino-5-morpholinomethyl-2-oxazolidinone

Abstract To detect furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) in fish sample, a Eu3+ labeling time-resolved fluoroimmunoassay (TRFIA) was developed. The experimental conditions were optimized as follows: the AMOZA-OVA concentration was 0.25 μg mL−1; the antibody was diluted by 50000 folds, and the competitive reaction time was 50 min. Under the optimal conditions, the method showed a detection limit of 0.01 ng mL−1, an IC50 of 0.26 ng mL−1 and a linear range (IC20−IC80) of 0.025−2.83 ng mL−1. The spiked recoveries of AMOZ in fish at three spiked levels ranged from 78.0% to 86.0%, and the relative standard deviations were less than 15%. Good correlation between ic-TRFIA and high performance liquid chromatography-tandem mass spectrometry was obtained for spiked food samples. The proposed ic-TRFIA method was suitable for the determination of AMOZ residue in food samples.

Determination of zearalenone with a glassy carbon electrode modified with nanocomposite consisting of palladium nanoparticles and a conductive polymeric ionic liquid

A glassy carbon electrode (GCE) modified with polymeric nanocomposite consisting of palladium nanoparticles and a conductive polymeric ionic liquid was prepared. The modified GCE was applied to sensitive and fairly selective electrochemical determination of the mycotoxin zearalenone. Electrocatalytic oxidation is performed in a solution containing 20 % (V/V) acetonitrile and 80 % (V/V) of 1 M perchloric acid. Cyclic voltammetry and square wave voltammetry revealed a well-defined electrocatalytic peak current at overpotential of +0.69 V versus Ag/AgCl. Under optimized experimental conditions, there is a linear relationship between anodic peak current and zearalenone concentration in the range from 0.03 to 35 ng⋅mL‾1, and the detection limit is 0.01 ng⋅mL‾1. The method was successfully applied to the analysis of zearalenone in spiked food samples and gave recoveries between 95.6 and 104.0 %.

SPE coupled to AAS trace determination of Cd(II) and Zn(II) in food samples using amine functionalized GMA-MMA-EGDMA terpolymer: Isotherm and kinetic studies

An ethylenediamine functionalized glycidyl methacrylate (GMA) based terpolymeric chelating resin was synthesized for the separation and preconcentration of Cd(II) and Zn(II) by SPE from bread, rice and fruit juice prior to FAAS determination. The resin was characterized by FT-IR, TGA/DTA, SEM, BET analysis and EDS. Synthesized resin shows a good capacity of 53.96mgg−1 for Cd(II) and 24.19mgg−1 for Zn(II) at pH 8.0. Five isotherm equilibrium models were studied to explain the sorption phenomenon out of which Langmuir, Dubinin–Radushkevich, Scatchard and Temkin models were found to be the best fitted. The limit of detection (LOD) and limit of quantification (LOQ) were observed to be 1.5 and 5.1μgL−1 for Cd and 1.2 and 4.1μgL−1 for Zn. The reliability of the method was investigated by the analysis of SRM and the recovery of analytes from various spiked food samples.

Development of a specific monoclonal antibody assay and a rapid testing strip for the detection of apramycin residues in food samples

ABSTRACTA specific monoclonal antibody (MAb) against apramycin (AP) was produced and used to develop an indirect competitive enzyme-linked immunosorbent assay (idcELISA) and a rapid testing strip for the detection of AP residues in foods. MAb exhibited negligible cross-reactivity with other aminoglycosides. Under optimized conditions in 0.01 M PBS, the half maximum inhibitory concentration (IC50) of MAb was 0.41 ng/ml with a limit of detection (LOD) of 0.15 ng/ml. The ELISA results were obtained within 90 min. The mean recoveries from all the spiked food samples were within the range of 79.02–105.49%, with coefficients of variation in the range of 2.21–11.4%. The strip test results obtained within 5 min had visual LODs in the range 2.5–5 µg/kg (ng/ml) for all food samples tested. Therefore, the developed strip test represents a fast and convenient detection method of AP residues in foods.

Simultaneous photometric microplate assay for free and total thiamine using gold nanoparticles and alkaline phosphatase

A photometric method is described for the determination of free and total thiamine (TH; vitamin B1) in food samples. It is based on the use of gold nanoparticles (AuNPs) which aggregate in the presence of TH owing to the interaction of its sulfur atom with the AuNPs. This is accompanied by a color change from wine red to purple-blue and an increase in absorbance at 590 nm. The effect is not observed for TH phosphates which, however, can be determined, as demonstrated for the example of TH pyrophosphate as a model ester, by treating them with alkaline phosphatase (ALP) which hydrolyses such esters. The use of two sample aliquots, one with and one without ALP, allows the determination of free and total TH, respectively. The dynamic range extends from 0.15 to 3.5 μM, and the detection limit is 54 nM of TH. The method has been applied to the analysis of spiked food samples and gave recoveries that ranged between 88.8 and 100.7 %.

Performance of the Assurance GDS® Assay for the Detection of L. monocytogenes in Pure Cultures and Spiked Food Samples

Listeria monocytogenes is a foodborne pathogen with significant impacts on public health and economy worldwide. Reliable and fast detection of L. monocytogenes is of major importance for both diagnostic laboratories and the food industry. The current study evaluated the performance of the Assurance GDS® assay for the detection of L. monocytogenes in pure cultures and spiked food samples. In the pure culture experiments, the Assurance GDS® assay for Listeria monocytogenes accurately detected the target strains of different serotypes and was correctly negative for a variety of other Listeria species. For reliable detection of L. monocytogenes in pure culture experiments, colony counts >105 cfu/ml were required, which emphasizes the need for an adequate enrichment step. The challenge test experiments (steak tartare, bologna type sausage, Gorgonzola cheese) using a one-broth enrichment strategy showed that the Assurance GDS® assay reliably detected L. monocytogenes after 16 h of enrichment in Half-Fraser broth, provided that spiking levels of the different matrices were ≥102 cfu/g. Depending of the food matrix, longer incubation times of 24 h or 48 h were required when the initial spiking level was <102 cfu/g, as to be expected in a proportion of naturally contaminated food products. Thus, the Assurance GDS® Listeria monocytogenes assay has proven to be a reliable and easy to handle, rapid test system for the specific detection of L. monocytogenes. This system is a suitable tool for generating microbiological results used for a “positive batch release”, especially for RTE foods with short shelf lives. However, longer enrichment times (24 h or 48 h) are required in a one-broth enrichment strategy, when the contamination level of the food matrix is low (<102 cfu/g).

A Monte Carlo approach to food density corrections in gamma spectroscopy

Evaluation of food products by gamma spectroscopy requires a correction for food density for many counting geometries and isotopes. An inexpensive method to develop these corrections has been developed by creating a detailed model of the HPGe crystal and counting geometry for the Monte Carlo transport code MCNP. The Monte Carlo code was then used to generate a series of efficiency curves for a wide range of sample densities. The method was validated by comparing the MCNP generated efficiency curves against those obtained from measurements of NIST traceable standards, and spiked food samples across a range of food densities.

Comparison of different methods for the recovery of DNA from spores of mycotoxin-producing moulds in spiked food samples.

Several food samples were spiked with fungal conidia to test the efficiency of different cell disruption methods and DNA extraction kits for subsequent molecular detection. For disrupting the firm cell walls of the spores, two different pretreatment methods, namely sonication and bead beating, were tested against no pretreatment. The subsequent DNA extraction and purification was performed using three different DNA extraction methods, which are based on a diverse combination of extraction principles, such as precipitation, thermic-enzymatic lysis, pH-enhancement and bonding with a silica membrane. The aim of the study was to find out the suitable pretreatment and DNA extraction method for the recovery of detectable amounts of fungal DNA from different food matrices. Significance and impact of the study: The choice of 'ready-to-use' commercial kits and methods has been of great importance regarding the recovery of extracted DNA. However, these commercially available kits are neither effective nor time-efficient when extracting DNA from fungal spores embedded in complex food matrices. Different extraction principles were compared and their effectiveness tested using real-time PCR. The combination of different principles for the extraction and purification of DNA was found as the most efficient method (quantity and purity) to obtain DNA from moulds and their spores from food samples.

Paper-based acetylcholinesterase inhibition assay combining a wet system for organophosphate and carbamate pesticides detection.

A dramatic increase in pesticide usage in agriculture highlights the need for on-site monitoring for public health and safety. Here, a paper-based sensor combined with a wet system was developed for the simple and rapid screening of organophosphate (OP) and carbamate (CM) pesticides based on the inhibition of acetylcholinesterase (AChE). The paper-based sensor was designed as a foldable device consisting of a cover and detection sheets pre-prepared with indoxyl acetate and AChE, respectively. The paper-based sensor requires only the incubation of a sample on the test zone for 10 minutes, followed by closing of the foldable sheet to initiate the enzymatic reaction. Importantly, the buffer loading hole was additionally designed on the cover sheet to facilitate the interaction of the coated substrate and the immobilized enzyme. This subsequently facilitates the mixing of indoxyl acetate with AChE, resulting in the improved analytical performance of the sensor. The absence or decrease in blue color produced by the AChE hydrolysis of indoxyl acetate can be observed in the presence of OPs and CMs. Under optimized conditions and using image analysis, the limit of detection (LOD) of carbofuran, dichlorvos, carbaryl, paraoxon, and pirimicarb are 0.003, 0.3, 0.5, 0.6, and 0.6 ppm, respectively. The assay could be applied to determine OP and CM residues in spiked food samples. Visual interpretation of the color signal was clearly observed at the concentration of 5 mg/kg. Furthermore, a self-contained sample pre-concentration approach greatly enhanced the detection sensitivity. The paper-based device developed here is low-cost, requires minimal reagents and is easy to handle. As such, it would be practically useful for pesticide screening by non-professional end-users.

Ultrasensitive determination of formaldehyde in environmental waters and food samples after derivatization and using silver nanoparticle assisted SERS

A selective and ultrasensitive surface-enhanced Raman spectroscopy (SERS) method was developed for the determination of formaldehyde (HCHO) in environmental waters and food samples. It is based on derivatization of HCHO with 4-amino-5-hydrazino-3-mercapto-1,2,4-triazole (AHMT) with HCHO. One of the products of the derivative reaction, 6-mercapto-5-triazolo[4,3-b]-s-tetrazine (MTT), can be quantified by SERS at 832 cm−1 using silver nanoparticles (AgNPs) as substrates. The incubation time for derivatization, the volume of reagents and the mixing times were optimized. The peak areas of the SERS are linearly related to the concentration of HCHO in the 1 − 1,000 μg L−1 range, the limit of detection is 0.15 μg L−1, and the limit of quantification is 0.45 μg L−1. Recoveries obtained by analyzing two spiked samples of environmental water and two spiked food samples were in the range between 91.0 and 108.7 %. There is no significant difference between the results obtained by the present method and the liquid chromatographic method (Chinese industrial standard method, SN/T 1547-2011).

Detection of the food allergen celery via loop-mediated isothermal amplification technique.

Since 2005, celery and celery products have to be labeled according to Directive 2003/89/EC due to their allergenic potential. In order to provide a DNA-based, rapid and simple detection method suitable for high-throughput analysis, a loop-mediated isothermal amplification (LAMP) assay for the detection of celery (Apium graveolens) was developed. The assay was tested for specificity for celery since closely related species also hold food relevance. The limit of detection (LOD) for spiked food samples was found to be as low as 7.8 mg of dry celery powder per kilogram. An evaluation of different amplification and detection platforms was performed to show reliable detection independent from the instrument used for amplification (thermal cycler or heating block) and detection mechanisms (real-time fluorescence detection, agarose gel electrophoresis or nucleic acid staining). The analysis of 10 commercial food samples representing diverse and complex food matrices, and a false-negative rate of 0% for approximately 24 target copies or 0.08 ng celery DNA for three selected food matrices show that LAMP has the potential to be used as an alternative strategy for the detection of allergenic celery. The performance of the developed LAMP assay turned out to be equal or superior to the best available PCR assay for the detection of celery in food products.

Development of a rapid and sensitive quantum dot-based immunochromatographic strip by double labeling PCR products for detection of Staphylococcus aureus in food

Staphylococcus aureus was listed as the 2nd common food-borne pathogens, resulting in diseases such as pneumonia, pseudomembranous colitis, pericarditis and even sepsis. A rapid, simple and sensitive detection method is required to monitor food in cases of contamination by S. aureus. Hence, a novel immunochromatographic test strip (ICTS), based on the two-component reaction (i.e. digoxigenin and anti-digoxigenin, quantum dots-conjugated streptavidin and biotin) in test line with QDs as indicator, was developed for the rapid and sensitive detection of S. aureus. Genomic DNA from bacteria lysis by boiling was extracted easily and rapidly with silica-coated magnetic nanoparticles, and a species-specific gene was amplified by PCR using digoxigenin/biotin-labeled primers. The fluorescence of captured QD labels on the test line and control line served as signals was observed by UV light. The sensitivity and specificity of the ICTS were estimated using bacteria spiked food samples (artificially mixed with cell counts (108 CFU/mL for each bacterium) of Escherichia coli, Listeria monocytogenes and Lactobacillus plantarum). The limit of detection for S. aureus was 3 × 100 CFU/mL and 3 × 101 CFU/g in spiked milk powder and meat samples, respectively, which was not affected by those non-S. aureus strains. Our results showed that QDs-based ICTS was promising for rapid and sensitive detection of S. aureus within 2 h. Hence, this protocol might be useful for screening and monitoring the contamination of S. aureus in food products, and helpful for promoting the prevention and control of communicable disease caused by food-borne pathogen.