Detection by multiplex real-time polymerase chain reaction assays and isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 in ground beef.

Six Shiga toxin-producing Escherichia coli (STEC) serogroups, which include O26, O45, O103, O111, O121, and O145, are responsible for the majority of non-O157 STEC infections in the United States, representing a growing public health concern. Cattle and other ruminants are reservoirs for these pathogens; thus, food of bovine origin may be a vehicle for infection with non-O157 STEC. Methods for detection of these pathogens in animal reservoirs and in food are needed to determine their prevalence and to develop intervention strategies. This study describes a method for detection of non-O157 STEC in ground beef, consisting of enrichment in modified tryptic soy broth at 42°C, followed by real-time multiplex polymerase chain reaction (PCR) assays targeting stx(1), stx(2), and genes in the O-antigen gene clusters of the six serogroups, [corrected] and then immunomagnetic separation (IMS) followed by plating onto Rainbow® Agar O157 and PCR assays for confirmation of isolates. All ground beef samples artificially inoculated with 1-2 and 10-20 CFU/25 g of ground beef consistently gave positive results for all of the target genes, including the internal amplification control using the multiplex real-time PCR assays after enrichment in modified tryptic soy broth for a total of 24 h (6 h at 37°C and 18 h at 42°C). The detection limit of the real-time multiplex PCR assays was ∼50 CFU per PCR. IMS for O26, O103, O111, and O145 was performed with commercially available magnetic beads, and the IMS beads for O45 and O121 were prepared using polyclonal antiserum against these serogroups. A large percentage of the presumptive colonies of each serogroup picked from Rainbow Agar O157 were confirmed as the respective serogroups; however, the percent recovery of STEC O111 was somewhat lower than that of the other serogroups. This work provides a method for detection and isolation in ground beef and potentially other foods of non-O157 STEC of major public health concern.

Development of a fluorescent latex microparticle immunoassay for the detection of staphylococcal enterotoxin B (SEB).

Staphylococcus aureus enterotoxin B (SEB) is a highly heat resistant enteric toxin with a potential as a biothreat agent. A sensitive method for the detection of staphylococcal enterotoxins is needed for food safety and food defense monitoring. The objectives of this research were to develop a competitive fluorescent immunoassay with detection of SEB below toxic levels of 1 ng/mL and to minimize sample preparation. Anti-SEB was immobilized onto carboxylated polystyrene microparticles, and SEB was labeled with fluorescein isothiocyanate (FITC). The concentrations of these reagents were optimized for the detection of SEB below 1 part per billion (1 ng/mL), and other assay conditions (sample volumes and incubation periods) were optimized. Drinking water and milk samples were spiked with 0.125-10 ng/mL SEB and were equilibrated overnight prior to analysis. The water and milk samples were directly analyzed, but heating the milk samples for 10 min at 90 degrees C improved the assay performance. SEB in samples bound with the anti-SEB linked to the latex followed by the competitive binding of SEB-FITC tracer. The excess, unbound tracer was separated by centrifugation, and the fluorescence density of the supernatant was measured. SEB was detected at levels as low as 0.125 ng/mL in drinking water and 0.5 ng/mL in whole milk. This fluorescent latex particle immunoassay will be utilized for the detection of SEB in various foods matrices.

Binding interaction studies of the immobilized Salmonella typhimurium with extracellular matrix and muscle proteins, and polysaccharides.

Our research attempts to understand the real-time interactions of immobilized Salmonella typhimurium with extracellular membrane proteins (collagen I, fibronectin and laminin) and muscle proteins (actin and myosin). Salmonella cells were immobilized on the sensor chip of a surface plasmon resonance (SPR) biosensor. Typical results showed that collagen I and myosin had higher binding responses to the S. typhimurium surface but laminin, actin and fibronectin had lower binding responses. The binding kinetics of collagen I and Salmonella cell surface showed an apparent dissociation and association rate constants of 3.90 E-4 s(-1) and 1.07 E+4 mol(-1) s(-1). Using the model system developed in our laboratory, the interactions of carrageenans and other polysaccharides with collagen and the Salmonella sensor surface were evaluated. The kappa-carrageenans blocked 92-100% binding of collagen to the Salmonella surface, while sodium alginate and low methoxy pectin blocked 50% and 18% binding, respectively. These biosensor studies allowed the rapid evaluation of compounds that may prevent bacterial attachment to poultry skin and carcasses, thus reducing pathogen contamination of poultry foods.

Development of a fluorescent latex immunoassay for detection of a spectinomycin antibiotic.

There is a need to develop a rapid and sensitive method to detect spectinomycin residues in animal tissues. A latex fluorescent immunoassay was designed using reagents developed for this assay. The spectinomycin antibody was produced in sheep, and the immunoglobulin (IgG) was purified through a Protein G affinity column and was immobilized onto latex particles. Spectinomycin was labeled with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein (DTAF). The optimum assay conditions consisted of preincubating the latex-IgG with spectinomycin in buffer solutions or in bovine kidney extracts. DTAF-spectinomycin was added and was further incubated. The bound spectinomycin-DTAF/IgG-latex complex was separated by centrifugation at 4000 g for 10 min. The fluorescence signals of the unbound spectinomycin-DTAF in the supernatant were measured at 485/535 nm excitation/emission. The measured signals were directly proportional to the concentration of spectinomycin in the samples, and spectinomycin was detected at 0-100 ppb with minimum detectability of 5 ppb. The mean regression correlation of four trials in buffer was 0.936 when the % bound complex vs spectinomycin concentration was plotted. Analysis of the kidney extract spiked with 0-100 ppb spectinomycin had a regression correlation of 0.959. This assay provides a rapid screening method for low ppb detection of spectinomycin.

Latex agglutination assays for detection of non-O157 Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145.

Latex agglutination assays utilizing polyclonal antibodies were developed for the top six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups. Rabbit antisera were affinity purified through protein A/G columns, and the isolated immunoglobulins (IgGs) were covalently immobilized onto polystyrene latex particles. The resulting latex-IgG complex had a protein (IgG) load of 0.20 to 0.28 mg/ml in a 1% latex suspension. Optimum conditions for the agglutination assay consisted of utilizing 20 µm l of latex-IgG reagent containing 2.0 to 2.8 µm g IgG in a 0.5% latex suspension. Agglutination or flocculation was observed almost instantly after mixing the colonies with the latex-IgG, indicating STEC strains. More than 100 target and nontarget strains were tested in more than 3,000 test replicates. All target organisms produced positive results, but three antisera (anti-O26, anti-O103, and anti-O145) cross-reacted with some other STECs. The anti-O103 and anti-O145 latex reagents cross-reacted with O26 strains, and the anti-O26 cross-reacted with O103 strains. The latex-IgG reagents are stable for at least 1 year and are easy to prepare. These agglutination assays can be used for identification of presumptive non-O157 STEC colonies from agar media. The techniques used to prepare the latex reagents also can be utilized for testing other STEC serogroups, other E. coli serotypes, or other pathogens to ensure safe foods to consumers.

Simple and rapid method for the analysis of phenolic compounds in beverages and grains.

A new method for the detection of phenolics in food systems was developed. This method is based on interactions of phenolics with Fast Blue BB diazonium salt in alkali pH, forming azo complexes, with the absorbance measured at 420 nm after 60 min. The linear regression correlations (R(2)) of gallic acid calibration standards were >0.99. The phenolic content (gallic acid equivalent) of samples analyzed yielded higher ratios (1.7-6.6) of the total phenolics by Fast Blue BB to Folin-Ciocalteu methods in most beverages and grain samples, but in flaxseed and some juice blends, the ratios were <1. The lower ratios suggest the presence of non-phenolic reducing constituents measured with the Folin-Ciocalteu method as "total phenolics". This method is simple and inexpensive and can be used to rapidly assess the total phenolics of foods and beverages.

Evaluation of Commercial Immunochemical Assays for Detection of Sulfamethazine in Milk.

Sulfamethazine (SMZ) is effective in the treatment of bacterial infections in food producing animals but its use is prohibited in dairy cows. Nevertheless, a 1988 survey of milk in ten cities conducted by the Food and Drug Administration revealed the presence of SMZ. Therefore, it was apparent that there was a need for rapid screening methods for SMZ. We evaluated commercial immunochemical test kits for SMZ with detectabilities of 1-10 parts per billion (ppb). Manipulations are suggested to effectively optimize immunochemical detection of SMZ in raw and processed fluid milk. The performances of the enzyme immunochemical test kits were evaluated by studying the effects of sample preparation, sample matrix, calibration and detection range of the kits using raw and processed milk samples. Immunochemical results were compared to quantitative high performance thin layer chromatography and high performance liquid chromatography with electrochemical detection. Both chromatographic methods had detectabilities in the low parts per billion range.