Rapid Detection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Food Using a Recombinase Polymerase Amplification-Based Assay

Staphylococcus aureus (S. aureus) is of great importance and is a leading cause of food poisoning, which is a public health concern in terms of the frequency and seriousness of the disease. In the present study, RPA and real-time RPA assays were developed and validated to detect S. aureus with high sensitivity and specificity by targeting the nuc gene for the first time. The analytical sensitivity of real-time RPA was 10² copies/reaction, which was higher than the sensitivity of the real-time PCR method. The analysis time was reduced to 10 min, but this method was as reliable as real-time PCR. Furthermore, the potential use of RPA to detect S. aureus was validated with five different artificially contaminated foods. In conclusion, the RPA and real-time RPA assays developed here, similar to real-time PCR, are rapid and simple and exhibit with high sensitivity and specificity. These assays serve as efficient tools for the detection of S. aureus in less advanced laboratories and are suitable for point-of-care detection.     

Saltatory Rolling Circle Amplification (SRCA): a Novel Nucleic Acid Isothermal Amplification Technique Applied for Rapid Detection of <Emphasis Type="Italic">Shigella</Emphasis> Spp. in Vegetable Salad

Shigella spp. are enteric pathogens that pose a serious threat to public health worldwide. A novel saltatory rolling circle amplification (SRCA) assay was developed to detect Shigella spp. in food targeting the ipaH gene. SRCA as an isothermal amplification method requires no expensive thermocycle instrument and could avoid electrophoresis as visualization results was successfully applied for SRCA. In order to confirm the specificity of this assay, 34 strains including 11 strains belonging to different Shigella species and 23 non-Shigella bacteria were detected with pure cultures. The sensitivity of Shigella flexneri by SRCA was evaluated using agarose gel electrophoresis, which was 7.3 × 10¹ fg/μL. In addition, the amplification results were also determined by adding the fluorochrome, SYBR Green I (1 μL of 1000×), allowing naked eye visualization of results, and the sensitivity was 7.3 × 10⁰ fg/μL. Moreover, the sensitivity of PCR was 7.3 × 10² fg/μL, showing that the sensitivity of SRCA by electrophoresis and SYBR Green I fluorescence were 10- and 100-fold higher than that of PCR, respectively. The detection limit of SRCA was also evaluated with artificially inoculated vegetable salad without enrichment, and it was 4.7 × 10² and 4.7 × 10¹ CFU/g by electrophoresis and fluorescence, respectively. The detection limit by PCR was 4.7 × 10³ CFU/g, which was 10- and 100-fold higher than that of SRCA. Therefore, SRCA is a potentially reliable tool for rapid and specific detection of Shigella in food and could be useful in underdeveloped countries with limited resources.     

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.     

Exposure assessment of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in <Emphasis Type="Italic">Kimbab</Emphasis>, a ready-to-eat Korean food

The exposure assessment was carried out for Staphylococcus aureus in kimbab by predicting growth of S. aureus and the production of enterotoxin using Food MicroModel® program. Environmental parameters selected were pH 5.5, Aw 0.999, and storage temperatures in the range of 10 to 30°C. It was predicted that 6.3 hr could be a critical time for enterotoxin production while kimbab was stored at 30°C. Mild case scenario analysis showed that enterotoxin could not be produced if kimbab was kept at 10°C during preparation and distribution and then left at 25°C for 4 hr before consumption. In the worst case scenario, the keeping time at 25°C was assumed to be 7.0 hr. The level of S. aureus in the worst case was predicted to be 6.8×10⁶ CFU/g which is lower than the critical level (7.8×10⁶ CFU/g) for toxin production.     

Detection of Viable <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Cells in Seafood Using a Real-Time Visual Loop-Mediated Isothermal Amplification Combined with Propidium Monoazide

Vibrio cholerae is an important foodborne pathogen causing severe intestinal infectious diseases that have high incidence and mortality. Almost all of rapid testing methods including immunological and molecular assays for V. cholerae are incapable of distinguishing live cells from dead ones, which may overestimate the number of bacteria and result in many false positive results. To address the problems, live cell-specific dye such as propidium monoazide (PMA) is employed. The loop-mediated isothermal amplification (LAMP) assay is a nucleic acid amplification method that is fast, specific, and sensitive. In this study, we developed a real-time visual LAMP assay using PMA dye to detect thyA gene, thereby identifying viable V. cholerae cells. The results showed that only V. cholarae strains could be detected, and there was no cross-reaction with non-V. cholarae strains. Besides, the sensitivity of the PMA-LAMP assay was 1.1 × 10² CFU/mL and the entire reaction could be accomplished within 1 h. The sensitivity was on par with that of the PMA-qPCR assay. The detection limit in different artificially inoculated samples was 5 CFU/25 g materials for the tested pathogens. In the practical test, the PMA-LAMP assay performed well in comparison with PMA-qPCR and the culture method. Hence, PMA-LAMP assay can provide a highly effective and rapid approach for detecting viable V. cholerae.     

Rapid Detection of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> in Shellfish by Real-Time Recombinase Polymerase Amplification

Vibrio parahaemolyticus (V. parahaemolyticus) is a zoonotic pathogen generally found in seafood. To detect the foodborne pathogen rapidly and accurately for food safety measures, we developed a real-time recombinase polymerase amplification (RPA) method. An evaluation of the specificity and sensitivity of the method is discussed here. A set of primers and probe was specially designed to target the tlh gene, which is usually regarded as a marker of total V. parahaemolyticus strains. During the reaction, target DNA was amplified and tagged with specific fluorophore within 10 min and at an incubation temperature of 40 °C. In addition to fast amplification and low temperature, the fluorescence signal was synchronized with the amplification of products for the generation of real-time data. The detection limit of this assay was 0.4 pg/μL of DNA, which is comparable to assays that use the bacterial culture as template, 4?×?10³ cfu/mL. The real-time RPA method had a stable performance when testing the spiking shellfish samples at the same level of contamination by the pathogen in different kinds of shellfish. Thus, the real-time RPA method shows great potential for on-site detection of V. parahaemolyticus, especially in low-resource settings.     

LAMP,PCR, and real-time PCR detection of <Emphasis Type="Italic">Acetobacter aceti</Emphasis> in yogurt

Acetic acid bacteria (AAB) can spoil food. Acetobacter aceti as a core subgroup of AAB is usually isolated from yogurt. A. aceti should be timely and effectively detected to prevent yogurt contamination. The present study focused on A. aceti to establish an assay that can be performed to detect AAB in yogurt. LAMP, PCR, and real-time PCR were applied and compared for detecting A. aceti from pure culture and artificially contaminated yogurt samples. In pure culture, LAMP showed the highest detection sensitivity with 10^?1 CFU/mL. For yogurt samples, the sensitivity limit of LAMP was 10² CFU/mL, which was lower than that of real-time PCR (10¹ CFU/mL). The results indicated that these methods could be quickly and efficiently applied to detect A. aceti. As LAMP technology has low cost and high detection efficiency, it can potentially be applied for detecting A. aceti in production and quality control programs of yogurt.     

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.     

Real-Time Recombinase Polymerase Amplification Assay for the Detection of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> in Seafood

Vibrio cholerae, the most hazardous Vibrio species, is threatening aquacultured animals and even human beings. In recent years, a few genetic markers that target V. cholerae have been reported, but their application was limited by techniques or themselves. The outer membrane lipoprotein gene lolB has been identified as a specific marker by using PCR and quantitative PCR methods. In this study, we developed a real-time recombinase polymerase amplification (RPA) assay targeting lolB gene in an attempt to provide a sensitive, rapid, and reliable detection method of V. cholerae. The sensitivity of RPA assay was determined by amplifying the standard plasmid dilutions. The detection limit down to five copies was achieved within 10 min, which was lower than that of qPCR (ten copies after 25 cycles within 1.5 h). The reproducibility of RPA assay was verified by eight independent experiments, presenting a high R ² value of the standard regression line (0.97). In addition, the specificity was confirmed with DNA extracted from other bacteria, shrimps, clams, and fishes using both methods. Finally, V. cholerae in shrimp samples were quantified using real-time RPA and qPCR with consistent outcomes, while no amplification was obtained from clam and fish samples using both methods. The applicability in the food industry was confirmed by quantitative detection of natural seafood.     

Quantification and Discrimination of Viable and Dead <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 Cells from Chicken Without Enrichment by Ethidium Bromide Monoazide Real-time Loop-Mediated Isothermal Amplification

In this study, a rapid and sensitive method of real-time loop-mediated isothermal amplification (Rti-LAMP) assays was developed for quantification and discrimination of viable and heat-killed E. coli O157:H7 cells treated with low concentration of ethidium bromide monoazide (EMA). Four micrograms per milliliter of EMA was chosen as the optimal concentration which did not inhibit DNA amplification derived from viable cells, but significantly increased the Tt values of dead cells in Rti-LAMP assays. When the DNA from 2.0?×?10³ viable CFU of E. coli O157:H7 was subjected to EMA-Rti-LAMP, the resulting Tt value was 17.73 min. In contrast, the DNA from 2.0?×?10³?CFU completely heat destroyed CFU of E. coli O157:H7 did not yield a positive amplification which Tt value was regarded as 60 min. When the DNA from viable plus heat-killed CFU at a ratio of 5:2995 was subjected to EMA-Rti-LAMP, the resulting Tt value was 23.06 min, which was statistically identical (P?<?0.05) to the Tt value of 24.07 min obtained with the DNA from only 5 viable CFU. The results indicate that even though 3.0?×?10³ dead cells yielded a negative amplification setting the Tt value as 60 min, low numbers of viable cells in the presence of much higher numbers of dead cells still yielded a linear plot for enumerating viable CFU from Tt values. Detection of E. coli O157:H7 derived from contaminated chicken samples, the EMA-Rti-LAMP could notably distinguish viable and heat-killed cells from 5.0?×?10¹ to 1.0?×?10⁴?CFU/g without enrichment.     

Quantitative Detection of <Emphasis Type="Italic">Clostridium perfringens</Emphasis> by Real-Time PCR in Raw Milk

Clostridium perfringens causes a broad spectrum of diseases in both humans and animals and is an important cause of foodborne illness. We developed and tested a real-time (Q-)PCR assay for the species-specific detection of C. perfringens that targets the phospholipase C (plc) gene and includes an internal amplification control (IAC), making it possible to identify false-negative results, which are common due to the high level of PCR inhibition by food compounds. The CPplc-IAC real-time PCR (RTi-PCR) assay was 100% selective, as shows with 36 Clostridium strains and 85 non-Clostridium strains, with an analytical sensitivity of 1 genome equivalent (GE) in 23% of the reactions and 10 GE in 100% of the reactions. The quantification was linear (R ² = 0.9990) over a 7-log dynamic range, down to 10 GE, with a PCR efficiency E = 0.841. The applicability of this RTi-PCR assay was assessed in milk samples. The assay detected as few as 300 spores in 25 mL of artificially contaminated raw sheep milk with 78% probability and 30 spores in 25 mL with 50% probability. It also has accuracy of 83.03 to 151.18%, as shown by an evaluation of the correspondence between RTi-PCR assay results and the number of spores per milliliter determinated by standard plating. This RTi-PCR method was effective for the detection and quantification of C. perfringens in milk having an important applicability in the control of this pathogen in the dairy food industry.     

Evaluation of <Emphasis Type="Italic">eryC</Emphasis> as a Molecular Marker for the Quantitative Detection of <Emphasis Type="Italic">Brucella</Emphasis> Spp. by Real-Time PCR in Food Samples

We evaluated the capacity of the Brucella sp. eryC gene as a diagnostic marker for brucellosis by quantitative real-time PCR. eryC gene encodes the enzyme d-erythrulose-1-phosphate dehydrogenase that plays an important role in the erythritol metabolism and is related with the Brucella survival in the intracellular environment of the macrophage. The assay includes an internal amplification control (IAC) in order to avoid false negative results. It was 100% specific, with an analytical sensitivity of 1 genome equivalent (GE) in 43% of the reactions, being the quantification highly linear (R ² > 0.9953) and efficient (PCR efficiency >0.8820) over a 6-log dynamic range, down to 10 GE. Finally, the applicability of this assay was evaluated with artificially contaminated biological matrices implicated in the transmission of this bacterium such as sheep raw milk and pig blood. The eryC-IAC real-time PCR assay allowed detection of as few as ten Brucella cells per 25 ml of sheep raw milk or per 1 ml of pig blood. In conclusion, we present an alternative for the detection of Brucella genus and therefore facilitate the establishment of preventive and prophylactic measures in food and farm environments.     

Application of the SureTect Detection Methods for <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Listeria</Emphasis> spp. in Meat,Dairy, Fish,and Vegetable Products

The aim of this study was to evaluate the application of the Listeria monocytogenes and Listeria spp. SureTect detection methods for a rapid and sensitive detection in an ample range of food products: raw pork and poultry meat, Spanish chorizo, pate, smoked salmon, raw sheep milk cured cheese, and ready-to-eat lettuce salad. The combination of a 24-h enrichment in the 24 Listeria Enrichment Broth (LEB) coupled to a rapid bacterial DNA extraction and real-time polymerase chain reaction (RTi-PCR) using the specific SureTect methods detected down to 2–6 L. monocytogenes CFU per sample in less than 27 h on the food categories tested. Furthermore, the applicability of L. monocytogenes and Listeria spp. SureTect detection methods in real samples was assessed using 303 food samples, obtaining at least the same analytical performance as the international reference method ISO 11290-1.     

Development of a Simple,Rapid Multiplex PCR Method Using the 16S rRNA Gene to Determine the Country of Origin of Brackish Water Bivalve <Emphasis Type="Italic">Corbicula japonica</Emphasis>

In this study, a multiplex PCR detection method was developed to identify the country of origin of Corbicula japonica (clams), a commercially important bivalve in Asia. Specific primer sets that have a single nucleotide mismatch at the 3′ terminus were designed after sequencing the mitochondrial 16S rRNA gene of clams identified as C. japonica originating from Korea, China, and Japan. Using this method, each origin was clearly identified based on the PCR products: three bands for Korean C. japonica (100, 283, and 384 bp), one band for Chinese C. japonica (384 bp), and two bands for Japanese C. japonica (384 and 100 bp). These results indicate that the 16S rRNA gene, which is usually used to identify species, can distinguish the country of origin within C. japonica. Our multiplex PCR assay should be a useful tool for the fair trade of the species.     

Development and characterization of reconstituted hydrogel from <Emphasis Type="Italic">Aloe vera</Emphasis> (<Emphasis Type="Italic">Aloe barbadensis</Emphasis> Miller) powder

Structural and rheological characterization of reconstituted hydrogels developed from A. vera non-fibrous alcohol insoluble residue (NFAIR) powder using different methods [viz., shaking (S), heating-shaking (HS), and heating (H)] and concentrations (viz., 0.2–1.6 %, w/v) was carried out. Functional group distribution by FTIR spectroscopy and Congo red (CR) method revealed the presence of acetylated acemannan in A. vera powder. Dynamic oscillation studies of A. vera (NFAIR) fluids at all concentrations of 0.2–1.6 %, w/v, showed gel strength in the order of H > HS > S method. However, in H method, increase in concentration from 0.2 to 1.6 %, w/v showed the conformational transition from semi-diluted solution to weak gel nature. Rheological models described the effect of heating temperatures (HT); 30–90 °C, and times (Ht); 15–60 min on viscoelastic behavior in reconstituted A. vera fluids. The reconstituted A. vera hydrogel prepared with a concentration of 1.6 %, w/v using 50 °C (HT) and 30 min (Ht) condition showed a good agreement with the Power law (storage modulus, G′) and Weak gel model (complex modulus, G*) fitted data (R² > 0.94) resulting higher viscoelastic moduli intercepts; G′₀ (71.5 Pa s ^n′), G″₀ (33.5 Pa s ^n″), lower slopes; n′ (0.22), n″ (0.06), higher network strength (A _F , 121.3 Pa s^1/z ) and number of network (z, 5.3) values. The obtained results suggested that heating at 50 °C/30 min can develop aqueous weak gel networks of A. vera with enhanced gel strength which may be utilized as a novel gelling agent for wide variety of targeted applications in food and pharmaceutical sectors.     

TLC-Digital Image-Based Fluorometric Analysis of Ergosterol and Chitin Content in Food Grains Artificially Infested with <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Fusarium verticillioides</Emphasis>

Novel thin-layer chromatography-digital image-based analytical methods were developed for the quantitation of ergosterol and chitin content in six food matrices (rice, wheat, maize, sorghum, groundnut, and sunflower), artificially infested with Aspergillus flavus (MTCC 6513)/Fusarium verticillioides (MRC 826). For ergosterol, single-step method, based on liquid/liquid extraction, was followed by thin-layer chromatography (TLC). Chitin was solubilized using lithium chloride (5%) in dimethyl acetamide and converted to chitosan using 5 N NaOH and subsequently complexed with calcofluor white dye. The absorption and emission maxima of chitosan-calcofluor complex were recorded at λ 350/230 and 430 nm, respectively. The sensitivity based on the limit of detection (LOD) was found to be 100 ng both for ergosterol and chitin analysis. Based on ergosterol and chitin analysis, groundnut and maize were found to be suitable substrates for A. flavus (p?<?0.013 and p?<?0.01), while sorghum followed by groundnut and sunflower were found to be ideal for F. verticillioides (p?<?0.01 and p?<?0.0001) and rice was established as poor substrate as there was no growth on it up to 12 days of incubation. A strong correlation was found between ergosterol and chitin contents with regression (r ²) values of 0.974 and 0.997 in food grains inoculated with A. flavus and F. verticillioides, respectively, during the period of infection. The authenticity of the two methods developed was further confirmed by applying them to commercial food grains and flours. Thus, ergosterol in combination with chitin analysis could be successfully used as an index of fungal contamination employing TLC-digital-based analytical methods.     

Effect of Tannin Extracts on Biofilms and Attachment of <Emphasis Type="Italic">Escherichia coli</Emphasis> on Lettuce Leaves

Lettuce is often involved in foodborne outbreaks caused by pathogenic Escherichia coli. Current control strategies have often proved ineffective to ensure safe food production. For that reason, the present study compared the efficacy of tannin extracts and chlorine treatments on the reduction of E. coli ATCC 25922 adhered to lettuce leaves. E. coli was inoculated artificially on leaf surfaces of fresh crisp lettuce. Effectiveness of water, chlorine (200 mg/L), and three commercial available tannin extracts from Acacia mearnsii De Wild. (tannin AQ (2 %, w/v), tannin SG (1 %, v/v) and tannin SM (1 %, v/v)) treatments was evaluated using the viable plate count method and scanning electron microscopy (SEM). SEM results revealed that bacterial cells are attached as individual cells and in clusters to the leaf surface after 2 h of incubation. Biofilm formation was observed after 24 h of incubation. The tannin SM treatment was able to reduce counts in approximately 2 log CFU/cm² on leaf segments. However, treatment was less effective in the reduction of E. coli counts after 24 h of incubation when compared to 2 h incubation of the same extract. The results suggest that the tannin SM extract diminishes E. coli counts adhered to and under biofilm formation on lettuce leaves and its effect is similar to the use of chlorine solutions.     

Real-time PCR systems for the detection of the gluten-containing cereals wheat,spelt, kamut,rye, barley and oat

Real-time PCR assays, using TaqMan^® probes, were applied to detect the gluten-containing cereals. Homologues target sequences encoding high molecular weight (HMW) glutenin were chosen to detect wheat, kamut, spelt and rye. For detecting barley, the gene Hor3 was selected. For the detection of oat the gene encoding the 12S seed storage protein was chosen. Based on this sequence data, primer and probe sequences were generated for the real-time PCR. A plant specific primer probe system based on 18S rRNA gene was chosen to detect amplificability of the extracted nucleic acids. The specificity of the primer and probe systems was checked using different lines from different origins of the species to be detected. The HMW glutenin system is specific for the corresponding species, as are the systems for the barley Hor3 gene and the oat 12S seed storage protein. The sensitivity of the systems was determined testing different matrices. With the HMW glutenin system 2.5 mg/kg of wheat in vegetable food matrices and 5 mg/kg of wheat in meat products were detected. The oat and the barley specific systems resulted in a sensitivity of 10 mg/kg. The detection method showed a satisfactory ruggedness.     

Use of Protein Hydrolysate from Yellow Stripe Trevally (<Emphasis Type="Italic">Selaroides leptolepis</Emphasis>) as Microbial Media

The objective of this study was to investigate the potential use of protein hydrolysate from yellow stripe trevally as a nitrogen source for the growth of different microorganisms. Protein hydrolysates from yellow stripe trevally with different degrees of hydrolysis (5, 15 and 25%) prepared using Alcalase (HA) or Flavourzyme (HF) were determined in comparison with commercial Bacto Peptone. For bacteria, Staphylococcus aureus and Escherichia coli, HF with 25% DH (HF₂₅) yielded the highest cell density and specific growth rate (μ _max) and the lowest generation time (t _d) (p?Saccharomyces cerevisiae and Candida albicans, Bacto Peptone yielded the higher growth rate than did HA and HF (p?μ _max and t _d were observed for fungus, Aspergillus oryzae (p?>?0.05). The pH of culture broth containing HF₂₅ decreased markedly during the first 8 hours of cultivation of S. aureus and E. coli (p?S. aureus (p?25 rendered the similar growth and colony size of S. aureus (p?>?0.05), compared with that containing Bacto Peptone. Scanning electron microscopic study revealed no differences in size and shape of microorganisms cultured in HF₂₅ and Bacto Peptone (p?>?0.05).     

A Rapid and Specific Biosensor for <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium Detection in Milk

This paper reports the application of an amperometric biosensor for rapid and specific Salmonella Typhimurium detection in milk. This device was developed from self-assembled monolayer technique on a gold screen-printed electrode, using cysteamine thiol. Polyclonal antibodies were oriented by protein A immobilization. The biosensor structure was characterized by cyclic voltammetry, Fourier transform infrared spectroscopy, and scanning electron microscopy. The analytical response was obtained by a chronoamperometry technique, using a direct-sandwich peroxidase-labeled system. The biosensor device showed a qualitative behavior with a very low limit of detection of 10 CFU mL^?1 and a detection time of 125 min. The biosensor specificity was demonstrated in pure and mixed samples with strains of Escherichia coli and Citrobacter freundii. The performance of the biosensor was found satisfactory, and the device was tested in skimmed and whole milk samples, being able to detect S. Typhimurium quickly, without an enrichment step. This structure of immunosensor assembly can be expended in future studies for other food matrices and bacterial species, making it a useful tool to ensure food safety.