Application of magnetic immuno-polymerase chain reaction assay for detection of <Emphasis Type="Italic">Salmonella</Emphasis> spp. in chicken meats

Since contaminated chicken meats have been the principal foodborne source of the contamination of Salmonella to human beings and cultural detection methods are labor-intensive and time-consuming, a study evaluating the performance of the combination of two techniques that are immunomagnetic separation (IMS) and polymerase chain reaction (PCR) for the detection of Salmonella in chicken meats was conducted. The IMS and PCR assay combines selective extraction of Salmonella by specific antibodies with primer-specific (primer pair based on the sequence of invA gene) PCR amplification. Initially chicken meat samples, in which no Salmonella contamination had been determined by using ISO 6579 reference method, were inoculated with Salmonella Enteritidis culture and subsequently the shortest non-selective pre-enrichment time, that had been needed for the detection of approximately 1 or 10 CFU/mL chicken meat levels of target bacteria by magnetic immuno-PCR assay, was found by using 14, 12, 10 and 8-h periods. In conclusion, it was found that magnetic immuno-PCR assay was able to detect 1–10 CFU Salmonella/25 g chicken meat, after only incorporating a non-selective pre-enrichment period of 12 h. Therefore, an overall 16-h (magnetic immuno-PCR assay in conjunction with 12-h non-selective pre-enrichment) magnetic immuno-PCR assay statistically evaluated as sufficient (p = 0.182 > 0.05) for rapid and sensitive detection of approximately 1–10 CFU Salmonella from 25 g chicken meat samples. Accordingly, 16-h magnetic immuno-PCR assay can be promising for routine use in the detection of Salmonella in chicken meat samples, and it consequently may prevent the risk of Salmonella infections in regard to chicken meats.     

Biotin exposure–based immunomagnetic separation coupled with sodium dodecyl sulfate,propidium monoazide,and multiplex real-time PCR for rapid detection of viable Salmonella Typhimurium,Staphylococcus aureus,and Listeria monocytogenes in milk

In this study, we established a rapid and sensitive method for the detection of viable Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes in milk using biotin-exposure-based immunomagnetic separation (IMS) combined with sodium dodecyl sulfate (SDS), propidium monoazide (PMA), and multiplex real-time PCR (mRT-PCR). We used IMS to lessen the assay time for isolation of target bacteria. We then optimized the coupling conditions and immunomagnetic capture process. The immunoreaction and incubation times for 5 μg of mAb coupled with 500 μg of streptavidin-functionalized magnetic beads using a streptavidin-biotin system were 90 and 30 min, respectively. Treatment with SDS-PMA before mRT-PCR amplification eliminated false-positive outcomes from dead bacteria and identified viable target bacteria with good sensitivity and specificity. The limit of detection of IMS combined with the SDS-PMA-mRT-PCR assay for the detection of viable Salmonella Typhimurium, Staph. aureus, and L. monocytogenes in spiked milk matrix samples was 10 cfu/mL and remained significant even in the appearance of 10⁶ cfu/mL of nontarget bacteria. The entire detection process was able to identify viable bacteria within 9 h. The combination of biotin-exposure-mediated IMS and SDS-PMA-mRT-PCR has potential value for the rapid and sensitive detection of foodborne pathogens.     

Vancomycin-modified poly-l-lysine magnetic separation combined with multiplex polymerase chain reaction assay for efficient detection of Bacillus cereus in milk

In this study, a new vancomycin (Van)-modified poly-l-lysine (PLL) magnetic bead (MB) technique was developed for isolation of gram-positive bacteria. The method combines magnetic separation with a multiplex PCR (mPCR) assay and gel electrophoresis for easy and rapid detection of Bacillus cereus. Vancomycin was used as a molecular ligand between the MB and the d-alanyl-d-alanine moieties on the cell wall surface of B. cereus. The PLL served as a flexible molecular tether between the MB and Van that reduced steric hindrance maintaining the biological activity of Van. The MB-PLL-Van capture nanoprobes exhibited excellent capture and isolation efficiency for B. cereus in spiked milk matrix samples without interference from the complex food matrix. The subsequent mPCR assay showed high specificity for the 4 target genes in B. cereus, the entFM, cesB, cer, and 16S rRNA genes, that were used to achieve efficient genotyping and detection. Under optimum conditions, the limit of detection reached 10³ cfu/mL, with a dynamic range of detection at 10³ to 10⁷ cfu/mL in pure culture. Application of the MB-PLL-Van mediated mPCR assay for B. cereus in milk matrix samples achieved results similar to those of the pure culture. In addition, with a 6-h pre-enrichment of B. cereus that was spiked in milk matrix samples, the limit of detection reached 10¹ cfu/mL. The MB-PLL-Van mediated mPCR assay developed in this study could be used as a universal technology platform for the efficient enrichment and genotyping of gram-positive bacteria.     

Multiplex PCR coupled with propidium monoazide for the detection of viable Cronobacter sakazakii,Bacillus cereus,and Salmonella spp. in milk and milk products

Cronobacter sakazakii, Bacillus cereus, and Salmonella spp. are common food-borne pathogens. The aim of this study was to develop a sensitive, specific, and rapid method for the simultaneous detection of these 3 pathogens in milk and milk products. Three specific primers were designed based on ompA, invA, and cesB of C. sakazakii, Salmonella spp. and B. cereus, respectively, for use in a multiplex PCR (mPCR). To eliminate false-positive results, cells were pretreated with propidium monoazide (PMA) for the selective elimination of the genomic DNA of dead cells. An internal amplification control was applied as an indicator of false-negative results from the interference of inhibitors in the food matrix. Results showed that, in pure culture, the limits of detection of the assay for C. sakazakii, Salmonella Enteritidis, and B. cereus were 9.5 × 10⁴, 7.4 × 10², and 7.5 × 10² cfu/mL, respectively. Moreover, 8 cfu/mL of viable B. cereus cells were detected after 5 h of enrichment, and 9 cfu/mL of viable C. sakazakii and 7 cfu/mL of Salmonella Enteritidis were detected after 7 h of enrichment in spiked pure milk, walnut peanut milk, and whole-wheat milk. To validate the PMA-mPCR assay, the PMA-mPCR assay and the traditional culture method were performed to detect the 3 bacterial strains in 1,165 milk product samples. The PMA-mPCR assay obtained the same results as the culture-based method. Results demonstrated that the PMA-mPCR assay has excellent sensitivity and specificity for the simultaneous detection of viable C. sakazakii, Salmonella Enteritidis, and B. cereus in milk and milk products.     

Rapid and simultaneous quantification of viable Escherichia coli O157:H7 and Salmonella spp. in milk through multiplex real-time PCR

Escherichia coli O157:H7 and Salmonella spp. in milk are 2 common pathogens that cause foodborne diseases. An accurate, rapid, specific method has been developed for the simultaneous detection of viable E. coli O157:H7 and Salmonella spp. in milk. Two specific genes, namely, fliC from E. coli O157:H7 and invA from Salmonella spp., were selected to design primers and probes. A combined treatment containing sodium deoxycholate (SDO) and propidium monoazide (PMA) was applied to detect viable E. coli O157:H7 and Salmonella spp. only. Traditional culture methods and SDO-PMA-multiplex real-time (mRT) PCR assay were applied to determine the number of viable E. coli O157:H7 and Salmonella spp. in cell suspensions with different proportions of dead cells. These methods revealed consistent findings regarding the detected viable cells. The detection limit of the SDO-PMA-mRT-PCR assay reached 10² cfu/mL for Salmonella spp. and 10² cfu/mL for E. coli O157:H7 in milk. The detection limit of SDO-PMA-mRT-PCR for E. coli O157:H7 and Salmonella spp. in milk was significantly similar even in the presence of 10⁶ cfu/mL of 2 nontarget bacteria. The proposed SDO-PMA-mRT-PCR assay is a potential approach for the accurate and sensitive detection of viable E. coli O157:H7 and Salmonella spp. in milk.     

Enrichment of injuredSalmonellain poultry products for detection by polymyxin–cloth enzyme immunoassay

Because injuredSalmonellacells have the potential to cause salmonellosis, their detection in foods is important to food safety testing programs. In pure cultures, a small number of heat-injuredSalmonella enteritidiscells were detected by the polymyxin–cloth enzyme immunoassay after 2-h static pre-enrichment in brain–heart infusion (BHI) medium supplemented with 0.05% yeast extract and 0.05% cholate (BYC), followed by overnight static enrichment in the presence of 0.3% selenite. However, in chicken carcass rinse, heat-injuredS. enteritidiscells (400 cfu ml^–1) could not be detected even after 6-h pre-enrichment in BYC, while uninjured cells were detectable. Prolonged (20 h) pre-enrichment in BYC, followed by 20-h incubation of a 10-fold dilution with BYC with addition of 0.03% selenite, permitted the detection of heat-injuredS. enteritidis(less than 7 cfu ml^–1) in the chicken carcass rinse.     

A simple and rapid realtime PCR assay for the detection of Shigella and Escherichia coli species in raw milk

Escherichia coli and Shigella spp. are two major bacterial contaminants in raw milk. Effective screening of the two microbes before starting the production process is a critical step for the quality and safety guarantee of the resulting dairy products. This study reported a rapid and simple realtime PCR assay using a ubiquitous primer and probe set targeting the tuf gene for the detection of E. coli and Shigella spp. An internal amplification control (IAC) was also comprised to indicate false-negative results. The duplex realtime PCR assay showed a high efficiency above 96 % and a detection limit <10 cfu per PCR. When artificially contaminated raw milk samples were further evaluated, the assay performed equally as well as the traditionally cultural-based method, and facilitated quantitative detection of the two microbes in the range from ~10² to ~10⁶ cfu mL^?1 raw milk. The detection limit was ~10² cfu mL^?1 raw milk for either or a mixture of the two strains without pre-enrichment step, and could be <10 cfu per 10 mL of raw milk if a pre-enrichment step was added. Considering the detection effectiveness, time-consumption saving, and economical efficiency, the present duplex realtime PCR assay has a great potential in the application in raw milk for assessing their microbiological quality and safety in relation to E. coli and Shigella spp.     

Rapid and sensitive detection of Salmonella in milk based on hybridization chain reaction and graphene oxide fluorescence platform

Salmonella is a foodborne pathogen that has contributed to numerous food safety accidents worldwide, making it necessary to detect contamination at an early stage. A pair of specific primers based on the invA gene of Salmonella was designed for PCR. Target double-stranded DNA (dsDNA) from PCR was purified and denatured at high temperature to obtain target single-stranded DNA (ssDNA). Two carboxyfluorescein-labeled hairpin probes (H1-FAM and H2-FAM) were designed with complementary portions to the ssDNA sequence so that binding could trigger H1-FAM and H2-FAM hybridization chain reaction (HCR) to produce a long dsDNA complex. In this study, graphene oxide (GO) was used in the development of a homogeneous fluorescence detection platform for Salmonella. Using this HCR-GO assay platform, Salmonella detection was completed in 3.5 h. Salmonella was reliably and specifically detected with a limit of detection (LOD) of 4.2 × 10¹ cfu/mL in pure culture. Moreover, this new HCR-GO assay platform was successfully applied to the detection of Salmonella in artificially contaminated milk with a LOD of 4.2 × 10² cfu/mL.     

Dual-Labeled PCR-Based Immunofluorescent Assay for the Rapid and Sensitive Detection of Enterotoxic <Emphasis Type="BoldItalic">Staphylococcus aureus</Emphasis> Using Cocktail-Sized Liposomal Nanovesicles as Signal Enhancer

Staphylococcus aureus is a major foodborne pathogen worldwide, and as little as 1 μg of staphylococcal enterotoxins (SEs) can cause food poisoning. Among SEs, enterotoxin A is the most common toxin that causes staphylococcal food poisoning. Hence, this work has developed a dual-labeled PCR-based immunofluorescent assay using anti-digoxigenin (DIG) antibody-tagged immunomagnetic beads (IMBs) as the capture reagent and cocktail-sized NeutrAvidin-tagged liposomal nanovesicles (NA-LNs) that encapsulate fluorescent dyes as the detection reagent. In this approach, the amplicon of sea gene was doubly labeled with DIG and biotin using modified primers and biotin-11-dUTP. The system depends on the immunocapture of IMB to pre-concentrate the labeled amplicons, which were further quantified using cocktail-sized NA-LNs, based on the release and subsequent measurement of encapsulated fluorescent dyes following the lysis of NA-LNs. After optimization, the developed assay could detect S. aureus and differentiate it from other common foodborne bacteria, such as Salmonella enterica and Escherichia coli, with a limit of detection (LOD) of 10¹ CFU mL^?1 without pre-enrichment. With a 2-h pre-enrichment, this developed assay could detect as little as 1 CFU in 25 mL of milk within a workday. Hence, this work established a rapid and sensitive PCR-based immunofluorescent assay using liposomal nanovesicles as an instant signal enhancer to detect the contamination of enterotoxic S. aureus in milk.     

Simultaneous quantitative detection of viable Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. using sodium deoxycholate-propidium monoazide with multiplex real-time PCR

Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. are common food-borne pathogens in milk that may cause serious diseases. In the present study, we established a simple, rapid, and specific method to simultaneously detect viable E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk. Three specific genes, fliC from E. coli O157:H7, cgcA from Cronobacter spp., and invA from Salmonella spp., were selected and used to design primers and probes. False-positive results were eliminated with the use of a combined sodium deoxycholate (SD) and propidium monoazide (PMA) treatment. Using the optimized parameters, this SD-PMA treatment combined with multiplex real-time PCR (SD-PMA-mRT-PCR) detected E. coli O157:H7, Cronobacter spp. and Salmonella spp. respectively, at 10² cfu/mL in pure culture or artificially spiked skim milk samples. A reasonable recovery rate (from 100 to 107%) for detection of viable bacteria using the SD-PMA-mRT-PCR assay was obtained in the presence of dead bacteria at 10⁷ cfu/mL. The SD-PMA-mRT-PCR method developed in this study can accurately detect and monitor combined contamination with E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk and milk products.     

Development of an isothermal amplification-based assay for the rapid visual detection of Salmonella bacteria

The efficient and timely detection of pathogens is a major concern worldwide. The aim of this study was to establish a rapid detection method for Salmonella bacteria in food samples to facilitate timely treatment. Widely used detection methods currently include culture-based methods and PCR-based methods. The former are time consuming, requiring 2 to 3 d, whereas the latter have higher accuracy but are typically complicated, requiring expertise and expensive instruments. In this study, a sensitive and rapid approach for the visual and point-of-use detection of Salmonella bacteria based on recombinase polymerase amplification (RPA) and a lateral-flow (LF) nucleic acid strip was established. We designed a pair of primers according to the invA gene of Salmonella bacteria: one was modified with digoxin, and the other was modified with biotin. In the presence of the biotin- and digoxin-modified primers and target DNA, the RPA produced a substantial amount of duplex DNA attached to biotin and digoxin. The products were detected using LF strips through immunoreaction: anti-digoxin antibodies on the gold nanoparticles, digoxin on the duplex, streptavidin on the LF test line, and biotin on the duplex. The developed RPA-LF assay allowed detection of Salmonella genomic DNA in less than 20 min with simple water bath equipment or portable thermal equipment. In addition, the RPA-LF assay was highly sensitive, with a detection limit as low as 20 fg of target DNA or 1.05 × 10¹ cfu of bacteria in pure culture, and highly specific, exhibiting no cross-reaction with Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Shigella, Enterobacter aerogenes, or Campylobacter jejuni. Importantly, Salmonella could be detected in milk and chicken breast at concentrations as low as 1.05 × 10⁰ cfu/mL or 1.05 × 10⁰ cfu/g after enrichment for 2 h and in eggs at 1.05 × 10⁰ cfu/g after enrichment for 4 h. Furthermore, RPA was more sensitive than PCR, which requires a thermal cycling device. In summary, this study describes a sensitive, simple, and point-of-use detection method for Salmonella bacteria.     

Short communication: Detection of stx2 and elt genes in bovine milk by using a multiplex PCR system

The aim of this study was to detect 2 important toxin genes from diarrheagenic Escherichia coli (DEC) in bovine milk using a new multiplex PCR. To standardize the multiplex PCR, the stx2 and elt genes were investigated for the detection of Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC), respectively. The DNA template was prepared with a thermal procedure (boiling) and a commercial kit. Samples consisted of UHT and pasteurized milk, both skimmed, and STEC and ETEC were tested in concentrations between 10¹ and 10⁹ cfu/mL. With the thermal procedure, the multiplex PCR system detected both pathotypes of E. coli at 10⁹ cfu/mL in UHT and pasteurized milk. When the commercial kit was used for template preparation, STEC and ETEC could be detected at concentrations as low as 10⁴ cfu/mL in UHT and pasteurized milk. Negative controls (Listeria monocytogenes, Salmonella Typhimurium, Salmonella Enteritidis, and Escherichia coli strain APEC 13) were not amplified with the multiplex PCR. These results indicate that the multiplex PCR was a rapid (less than 6 h) and efficient method to detect STEC and ETEC in milk using different methods for DNA preparation; however, the commercial kit was more sensitive than the thermal procedure.     

Detection of Salmonella in food over 30 h using enrichment and polymerase chain reaction

A 30-h method for the detection of Salmonella spp. in food was developed. The method involved preenrichment in buffered peptone water for 6–8 h, immunomagnetic separation (IMS) using Dynabeads^®anti-Salmonella , selective enrichment in Rappaport-Vassiliadis broth for 16–18 h, lysis of bacterial cells in sodium dodecylsulfate and NaOH solution at 95°C, and the polymerase chain reaction (PCR) using primers ST11 and ST15. The detection limit of the method was 10⁰cfu 25g⁻¹, as determined by the analysis of food samples artificially contaminated with S. enteritidis. When the reference material containing on average 5 cfu of S. panama was used for the artificial contamination, 3 out of 22 food samples were found to be false-negative. When the method was evaluated in comparison with the standard ISO 6579 method on 42 possibly naturally-contaminated food samples, one sample was found positive by the 30-h method, one sample was found positive by the ISO-method, and two samples were found positive by both methods. The developed method proved rapid, but produced a non-zero level of false-negative results.     

Two-step large-volume magnetic separation combined with PCR assay for sensitive detection of Listeria monocytogenes in pasteurized milk

Immunomagnetic separation (IMS) is an effective tool for the preconcentration and purification of food-borne pathogens from complex food samples because of its high capture efficiency (CE). In conventional IMS, antibodies are usually conjugated on the surface of magnetic beads (MB); the random orientation and conformation changes of antibodies on the MB surface can decrease their bioactivity. Moreover, the Brownian motion of immobilized antibodies is weakened, thereby rendering their binding efficiency with bacteria lower than that of free antibodies. Thus, abundant antibodies are commonly required to ensure high CE for IMS, particularly for large volumes. In this study, a 2-step large-volume magnetic separation (10 mL) was proposed to preconcentrate Listeria monocytogenes from pasteurized milk. First, the biotinylated anti-L. monocytogenes monoclonal antibodies (mAb) were bound with L. monocytogenes in 10 mL of diluted milk through an antigen-antibody interaction, and then streptavidin-labeled MB were used to capture biotin-mAb coated with L. monocytogenes by biotin and streptavidin interaction. Under optimal conditions, the CE of 2-step magnetic separation was >90% with L. monocytogenes concentrations ranging from 8 × 10⁰ to 8 × 10⁴ cfu/mL, whereas the amount of biotin-mAb was 14 fold lower than that of the conventional IMS method. Coupled with a PCR assay, the detection limit of the proposed method was 8 × 10⁰ cfu/mL in pure culture and 8 × 10¹ cfu/mL in pasteurized milk without any pre-enrichment process. Moreover, the overall detection time, including sample preparation, large-volume magnetic separation, and PCR, took less than 7 h. In summary, the developed 2-step large-volume IMS combined with PCR was highly sensitive and low cost and, thus, has considerable potential for the rapid screening of food-borne pathogenic bacteria.     

Poly-l-lysine-functionalized magnetic beads combined with polymerase chain reaction for the detection of Staphylococcus aureus and Escherichia coli O157:H7 in milk

Rapid and credible detection of pathogens is essential to prevent and control outbreaks of foodborne diseases. In this study, a poly-l-lysine-functionalized magnetic beads (PLL-MB) strategy combined with a PCR assay was established to detect Staphylococcus aureus. We also detected Escherichia coli O157:H7 to further verify the strategy for gram-negative bacteria detection. Poly-l-lysine has strong positive charges because of its amino groups, which can conjugate with the carboxyl of carboxyl magnetic beads. Furthermore, it can be used to combine with bacteria through electrostatic adsorption. Under optimum conditions, the developed PLL-MB complexes showed 90% capture efficiency in phosphate-buffered saline and 85% capture efficiency in milk for S. aureus detection. The limit of detection of the PLL-MB-PCR assay was 10² cfu/mL (1.8 × 10² cfu/mL for S. aureus and 7 × 10² cfu/mL for E. coli O157:H7) in phosphate-buffered saline and milk samples. The whole assay can be performed within 4 h. The proposed strategy showed a lower limit of detection when compared with the conventional PCR assay without enrichment. In addition, this method exhibited the advantages of a high-efficient, cost-efficient, and simple operation, indicating its potential applications in foodborne pathogen detection.     

The detection of Salmonella serovars from animal feed and raw chicken using a combined immunomagnetic separation and ELISA method

The immunomagnetic separation technique was used in conjunction with an ELISA-based detection system to recover Salmonella enteritidis, Salmonella typhimurium and Salmonella virchow (2–2×10³cfu 25 g⁻¹) from raw chicken and animal feed. The detection time was less than 27 h and included the conventional pre-enrichment (18 h) step in buffered peptone water and an enrichment step (6 h) in glucose nutrient broth. In contrast, the conventional method using Rappaport-Vassiliades enrichment broth and selective plating on XLD agar required 72–96 h.     

A fluorescent biosensor based on quantum dot–labeled streptavidin and poly-l-lysine for the rapid detection of Salmonella in milk

Salmonella, as a common foodborne pathogen in dairy products, poses a great threat to human health. We studied a new detection method based on quantum dots (QD). A fluorescent biosensor with multiple fluorescent signal amplification based on a streptavidin (SA) biotin system and the polyamino linear polymer poly-l-lysine (PLL) were established to detect Salmonella in milk. First, Salmonella was captured on a black 96-well plate with paired Salmonella mAb to form a double-antibody sandwich. Second, SA was immobilized on biotin-modified mAb by SA-biotin specific bond. Then, the biotin-modified polylysine (BT-PLL) was bound on SA and specifically bonded again through the SA-biotin system. Finally, water-soluble CdSe/ZnS QD-labeled SA was added to a black 96-well plate for covalent coupling with BT-PLL. The fluorescent signal was amplified in a dendritic manner by the layer-by-layer overlap of SA and biotin and the covalent coupling of biotinylated PLL. Under optimal conditions, the detection limit was 4.9 × 10³ cfu/mL in PBS. The detection limit was 10 times better than that of the conventional sandwich ELISA. In addition, the proposed biosensor was well specific and could be used for detecting Salmonella in milk samples.     

Rapid and reliable identification of Staphylococcus aureus harbouring the enterotoxin gene cluster (egc) and quantitative detection in raw milk by real time PCR

A TaqMan and a SYBR Green real time PCR (rt-PCR) were developed for the reliable identification and quantitative detection of Staphylococcus (S.) aureus strains harbouring the enterotoxin gene cluster (egc) regardless of its variants. Both approaches revealed 100% specificity against a panel of 70 reference strains, including 29 clinical and foodborne S. aureus strains harbouring all the egc variants to date known, 4 egc⁻S. aureus strains and 37 strains of phylogenetically closely and distantly related species. Standard curves made by 10 fold dilutions of either genomic DNA or cells from an egc⁺S. aureus log-phase broth culture showed a good linearity of response (R² ≥ 0.993) for six orders of magnitude, with about 100% relative accuracy and a low inter-assay variability (CV ≤ 3.02). The overall limit of quantification (LOQ) for both rt-PCR assays (about 100% PCR efficiency; running time 30 min) was 10 cfu or 10 genome equivalents per reaction mixture although 1 cfu or 1 genome equivalent was detected with a 33.33% probability. These performances were confirmed in raw milk artificially contaminated with log-phase broth cultures of either a single egc⁺S. aureus strain or a mixture of S. aureus strains harbouring all the egc variants to date known. Similar results were also obtained with a raw milk based standard curve of the S. aureus egc⁺ mixture in the presence of 10⁶ cfu/mL of egc⁻S. aureus strains harbouring some of the commonest enterotoxin genes associated to the staphylococcal food poisoning. Nonetheless, the TaqMan based approach resulted in a lower sensitivity (LOQ = 100 cfu equivalents per reaction mixture) than the SYBR Green based assay (LOQ = 10 cfu equivalents per reaction mixture). When applied to real milk samples, both PCR assays provided a good response with 100% diagnostic specificity and 96-107% relative accuracy, as compared to conventional culture-based PCR approaches. Due to the high specificity, the wide dynamic range of detection and the high sensitivity demonstrated even in a complex and potentially highly contaminated raw milk matrix, the SYBR Green rt-PCR assay is a useful diagnostic tool for quick, high throughput and reliable routine screening of egc⁺S. aureus isolates. Moreover, the SYBR Green based quantitative detection of these pathogens in raw milk could remarkably contribute to clarify their actual role in staphylococcal food poisoning and other clinical syndromes associated with the consumption of milk and milk-based products.     

A novel electrochemical immunosensor based on Fe3O4@graphene nanocomposite modified glassy carbon electrode for rapid detection of Salmonella in milk

Foods contaminated by foodborne pathogens have always been a great threat to human life. Herein, we constructed an electrochemical immunosensor for Salmonella detection by using a Fe₃O₄@graphene modified electrode. Because of the excellent electrical conductivity and mechanical stability of graphene and the large specific surface area of Fe₃O₄, the Fe₃O₄@graphene nanocomposite exhibits an excellent electrical signal, which greatly increased the sensitivity of the immunosensor. Gold nanoparticles were deposited on Fe₃O₄@graphene nanocomposite by electrochemical technology for the immobilization of the antibody. Cyclic voltammetry was selected to electrochemically characterize the construction process of immunosensors. The microstructure and morphology of related nanocomposites were analyzed by scanning electron microscopy. Under optimized experimental conditions, a good linear relationship was achieved in the Salmonella concentration range of 2.4 × 10² to 2.4 × 10⁷ cfu/mL, and the limit of detection of the immunosensor was 2.4 × 10² cfu/mL. Additionally, the constructed immunosensor exhibited acceptable selectivity, reproducibility, and stability and provides a new reference for detecting pathogenic bacteria in milk.