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Under favorable growth conditions, Staphylococcus aureus infects and causes illnesses in humans and animals. Staphylococcal enterotoxins are considered the most important factor for food poisoning, with these toxins usually found in milk and its byproducts. This study investigated the presence of a classical enterotoxin of S. aureus (enterotoxin A) in raw cow milk samples obtained from Region 3 of Tehran. Sixty cow milk samples were collected from traditional dairy sales centers in the study site. Electrophoresis, high-performance liquid chromatography, sandwich ELISA, and direct ELISA were evaluated as methods for detecting toxins in the milk. A direct ELISA kit was designed, with the best factors, the most suitable densities, and the most optimal temperatures and temporal incubation conditions incorporated into the design. Among the evaluated methods, direct ELISA exhibited the highest accuracy in toxin detection. The designed kit achieved a correctness coefficient of 0.98 and detected enterotoxin A in 23% of the samples. It also exhibited a relative sensitivity and other features that are comparable to those of other bacterial detection techniques. Finally, the kit achieved good matching and repetition results, and no cross-reactions occurred between enterotoxins during the procedure., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the present study, a sensitive and specific IgY mediated ImmunoCapture-PCR-ELISA (IC-PCR-ELISA) was developed for the detection of staphylococcal enterotoxin A (SEA) from culture supernatants and suspected contaminated samples. Due to the virtue of avian immunoglobulins (IgY) to have the least affinity towards staphylococcal protein A (SpA) responsible for false positives, we employed anti-SEA IgY for capture of SEA toxin and revealed with SEA specific rabbit antibodies conjugated to a 524bp DNA marker. Biotin-11-dUTP was incorporated during PCR amplification and post PCR analysis was performed by PCR-ELISA. Unlike IgG immunocapture, IgY mediated immunocapture of SEA was free from false positives due to protein A. The developed assay was specific to SEA except for minor cross reactivity with staphylococcal enterotoxin E (SEE). Several raw milk samples were evaluated for the presence of SEA with and without enrichment. Three samples were found to be positive for SEA after enrichment for 8h. Though IC-PCR-ELISA for SEA showed 100% correlation with PCR analysis for sea gene, the assay was unique in terms of sensitivity of detecting ~10pg/ml of SEA toxin from spiked milk samples. Result of IC-PCR-ELISA was further confirmed by conventional methods of isolation and characterization. The presented method can be very useful for rapid analysis of milk samples for SEA contamination and can be further extended for detection of multiple SE's in different wells of same PCR plate using common DNA substrate., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Staphylococcus aureus (S. aureus) is one of the most important microorganisms causing foodborne outbreaks worldwide. the present work was planned to study the occurrence, count, and identification of S. aureus and investigation of the obtained isolates for the presence of enterotoxin A encoding gene (sea) in milk and some milk products in New Valley governorate, Egypt. A total of 150 random samples of marketable milk and some milk products including soft cheese (Kareish, Domiati) & ice cream (30 for each) were collected from different localities in the New Valley governorate. S. aureus occurred in 11.6, 10, 3.3 and10% for marketable milk, Kariesh cheese, Domiati cheese, and ice cream, respectively. The presence of the enterotoxin sea gene was identified in 4 (57%) and 2 (50%) in milk and soft cheese, respectively, while none of the isolates obtained from ice cream samples harbored this gene. The investigation revealed that the examined milk samples and its products were contaminated with S. aureus with average counts of 2.8×108, 7.5×105, 7.6×106, and 1.7×107 for milk, Kariesh cheese, Domiati cheese, and ice cream samples, respectively. In conclusion, the presence of S. aureus in milk and some milk products indicated that the examined samples were of low quality which reflects unhygienic measures during milking, processing, storage, and distribution chain. [ABSTRACT FROM AUTHOR]

Staphylococcal enterotoxin A (SEA) is an exotoxin that releases from gram-positive bacteria, and it is a source of food poisoning. Here, the luminescence method is introduced to quantify and detect SEA using carbon quantum dots (CQDs) as fluorophore; SEA antibody loaded Fe3O4 nanoparticles (Fe3O4@Ab) are prepared for SEA extraction. SEA in standards and milk samples were conjugated with Fe3O4@Ab (Fe3O4@Ab@SEA), separated by a 1.4 T magnet, and dispersed in phosphate buffer saline. The fluorescence of CQDs is quenched when mixed with Fe3O4@Ab@SEA, without elution of SEAs from the Fe3O4@Ab@SEA. It is a fast and reliable method compared with the immunoassay counterpart. At optimum quantification conditions, a linear relationship between SEAs concentration and CQDs fluorescent quenching was observed from 4 to 2000 pg mL−1 with the detection limit 2 pg mL−1. Repeatability and reproducibility were calculated as a percentage of relative standard deviations and were from 1.56 to 11.76%. Finally, the proposed method was applied to measure the SEA contents of four sterilized and homogenized milk samples without fat precipitations and have shown significant reliability. [ABSTRACT FROM AUTHOR]

Ochratoxin A (OTA) and staphylococcus enterotoxin A (SEA) are highly toxic contaminants and have induced human health problems. They commonly occur in milk and milk products. A competitive fluorescent immunoassay was developed for rapid and simultaneous determination of these toxins in milk samples. The procedure was based on the competitive immunoreactions between antigens in sample and antigen-fluorescent dye conjugates with immobilised antibodies on magnetic nanoparticles (MNPs). Each monoclonal antibody specifically recognises its corresponding toxin (antigen), and there is no cross-reactivity in the assay. First, monoclonal antibodies against OTA and SEA were produced. The activity of the obtained antibodies was determined by fluorescent-linked immunosorbent assay. Then, the monoclonal antibodies were immobilised on MNPs. The amounts of immobilised anti-OTA antibody and anti-SEA antibody were determined to be 20 and 22 μg mL−1, respectively. The antigen-fluorescent dye conjugates OTA–OVA–ATTO620 and SEA-FITC were prepared. The optimal amount of immobilised antibodies for competitive immunoassay was determined. It was found that the linear range of OTA in buffer was larger (0.001–100 ng mL−1) than the linear range of SEA (0.001–20 ng mL−1). The results for simultaneous determination of OTA and SEA in sixfold diluted milk were almost the same in buffer; the linear range for OTA was from 0.005 to 100 ng mL−1 and for SEA from 0.005 to 20 ng mL−1. The detection limit for both OTA and SEA in milk was 0.004 ng mL−1. The developed method took half the time of the individual assays (20 min). The assay was evaluated using spiked milk samples. The influences of somatic cell count, fat, pH and protein concentration in milk on immunoassay were studied. In summary, this developed immunoassay could provide an effective and rapid approach for detecting multi-toxins in milk samples. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a potential pathogen contaminating raw milk and dairy products, where it is able to produce thermostable enterotoxins that can cause staphylococcal food poisoning. This study was undertaken to investigate the inhibitory activity of a Lactococcus lactis strain (isolated from milk) on S. aureus growth and staphylococcal enterotoxin A (SEA) production. In the presence of L. lactis, the number of the pathogen decreased significantly (p

Staphylococcus aureus is closely associated with food poisoning incidences, more, because of intoxication due to contamination of food with its enterotoxins. Staphylococcus enterotoxin A (SEA) is highly resistant to heat and other food processing conditions and has been implicated in food borne diseases from dairy products. Here we describe a rapid, gold nanoparticle based lateral flow immunoassay (LFIA) for detecting SEA in milk. Assay is in sandwich format and uses two specific monoclonal antibodies against SEA, one of which is labeled with gold nanoparticles and other is immobilized at control line. We also demonstrate the effect of using a combination of additives in capture antibody in order to enhance assay sensitivity. The developed LFIA is specific and has a lower detection limit of 0.5 μg/ml. LFIA is tested both on spiked buffer and milk samples and display good reproducibility. The assembled strips stable for a month at 4 °C. The method may be suitable for testing SEA in milk samples as it is quick, easy to perform and reproducible. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a potential pathogen contaminating raw milk and dairy products, where it is able to produce thermostable enterotoxins that can cause staphylococcal food poisoning. This study was undertaken to investigate the inhibitory activity of a Lactococcus lactis strain (isolated from milk) on S. aureus growth and staphylococcal enterotoxin A (SEA) production. In the presence of L. lactis, the number of the pathogen decreased significantly (p<0.05) after 6 h of incubation in a laboratory medium and milk (3 log CFU/mL reduction compared to pure cultures). SEA concentration was reduced by 79 % in the co-cultures. S. aureus was unable to reach population levels permitting SEA production in the cheese inoculated with L. lactis during 32 days of storage. In contrast, during the same period, it attained 7 log CFU/g in the cheese manufactured without the lactococcal strain, a level which permitted SEA detection in the cheese extracts. However, this enterotoxin was never detected in the cheese harbouring L. lactis. These results demonstrate the anti-staphylococcal enterotoxinogenesis potential of the L. lactis strain and its usefulness in raw milk cheese biopreservation. [ABSTRACT FROM AUTHOR]

A simple, cheap, and less aggressive immobilization procedure for biomolecules using reduced graphene oxide (rGO) was employed to prepare an impedimetric immunosensor for detection of staphylococcal enterotoxin A (SEA) from Staphylococcus aureus in milk samples. The scanning electron microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy (EIS) were used to monitor the single steps of the electrode assembly process. The glassy carbon (GC)/rGO platform detected the antigen-antibody binding procedures of SEA with concentrations of 0.5 to 3.5 mg L−1 via impedance changes in a low frequency range. The impedimetric immunosensor was successfully applied for the determination of SEA in milk samples. [ABSTRACT FROM AUTHOR]