Studies on Simultaneous Enrichment and Detection of Escherichia coli O157:H7 during Sample Shipment.

The USDA-FSIS has zero tolerance for E. coli O157:H7 in raw ground beef. Currently, FSIS collects samples from beef processing facilities and ships them overnight to regional testing laboratories. Pathogen detection requires robust methods that employ an initial 15-24 h culture enrichment. This study assessed the potential of using the ΦV10nluc phage-based luminescence detection assay during enrichment while the sample is in transit. Parameters including phage concentrations, temperature, and media-to-sample ratios were evaluated. Results in liquid media showed that 1.73× 103 pfu/mL of ΦV10nluc was able to detect 2 CFU in 10 h. The detection of E. coli O157:H7 was further evaluated in kinetic studies using ratios of 1:3, 1:2, and 1:1 ground beef sample to enrichment media, yielding positive results for as little as 2-3 CFU in 325 g ground beef in about 15 h at 37 °C. These results suggest that this approach is feasible, allowing the detection of a presumptive positive upon arrival of the sample to the testing lab. As the current cargo hold controlled temperature is required to be 15-25 °C, the need for elevated temperature should be easily addressed. If successful, this approach could be expanded to other pathogens and foods.

Flow-Through Electrochemical Biosensor for the Detection of Listeria monocytogenes Using Oligonucleotides.

Consumption of food contaminated by Listeria monocytogenes can result in Listeriosis, an illness with hospitalization rates of 94% and mortality rates up to 30%. As a result, U.S. regulatory agencies governing food safety retain zero-tolerance policies for L. monocytogenes. However, detection at such low concentrations often requires strategies such as increasing sample size or culture enrichment. A novel flow-through immunoelectrochemical biosensor has been developed for Escherichia coli O157:H7 detection in 1 L volumes without enrichment. The current work further augments this biosensor's capabilities to (1) include detection of L. monocytogenes and (2) accommodate genetic detection to help overcome limitations based upon antibody availability and address specificity errors in phenotypic assays. Herein, the conjugation scheme for oligo attachment and the conditions necessary for genetic detection are laid forth while results of the present study demonstrate the sensor's ability to distinguish L. monocytogenes DNA from L. innocua with a limit of detection of ~2 × 104 cells/mL, which agrees with prior studies. Total time for this assay can be constrained to <2.5 h because a timely culture enrichment period is not necessary. Furthermore, the electrochemical detection assay can be performed with hand-held electronics, allowing this platform to be adopted for near-line monitoring systems.

Advances in Foodborne Pathogen Analysis.

As the world population has grown, new demands on the production of foods have been met by increased efficiencies in production, from planting and harvesting to processing, packaging and distribution to retail locations. These efficiencies enable rapid intranational and global dissemination of foods, providing longer "face time" for products on retail shelves and allowing consumers to make healthy dietary choices year-round. However, our food production capabilities have outpaced the capacity of traditional detection methods to ensure our foods are safe. Traditional methods for culture-based detection and characterization of microorganisms are time-, labor- and, in some instances, space- and infrastructure-intensive, and are therefore not compatible with current (or future) production and processing realities. New and versatile detection methods requiring fewer overall resources (time, labor, space, equipment, cost, etc.) are needed to transform the throughput and safety dimensions of the food industry. Access to new, user-friendly, and point-of-care testing technologies may help expand the use and ease of testing, allowing stakeholders to leverage the data obtained to reduce their operating risk and health risks to the public. The papers in this Special Issue on "Advances in Foodborne Pathogen Analysis" address critical issues in rapid pathogen analysis, including preanalytical sample preparation, portable and field-capable test methods, the prevalence of antibiotic resistance in zoonotic pathogens and non-bacterial pathogens, such as viruses and protozoa.

Impacts of Clarification Techniques on Sample Constituents and Pathogen Retention.

Determination of the microbial content in foods is important, not only for safe consumption, but also for food quality, value, and yield. A variety of molecular techniques are currently available for both identification and quantification of microbial content within samples; however, their success is often contingent upon proper sample preparation when the subject of investigation is a complex mixture of components such as foods. Because of the importance of sample preparation, the present study employs a systematic approach to compare the effects of four different separation techniques (glass wool, 50 µm polypropylene filters, graphite felt, and continuous flow centrifugation (CFC)) on sample preparation. To define the physical effects associated with the use of these separation methods, a multifactorial analysis was performed where particle size and composition, both pre- and post- processing, were analyzed for four different food matrices including lean ground beef, ground pork, ground turkey and spinach. Retention of three important foodborne bacterial pathogens (Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes) was also examined to evaluate the feasibility of the aforementioned methods to be utilized within the context of foodborne pathogen detection. Data from the multifactorial analysis not only delineated the particle size ranges but also defined the unique compositional profiles and quantified the bacterial retention. The three filtration membranes allowed for the passage of bacteria with minimal loss while CFC concentrated the inoculated bacteria. In addition, the deposition and therefore concentration of food matrix observed with CFC was considerably higher for meat samples relative to spinach. However, filtration with glass wool prior to CFC helped clarify meat samples, which led to considerably lower amounts of solids in the CFC vessel post processing and an increase in the recovery of the bacteria. Overall, by laying a framework for the deductive selection of sample preparation techniques, the results of the study can be applied to a range of applications where it would be beneficial to scientifically guide the pairing of the criteria associated with a downstream detection method with the most advantageous sample preparation techniques for complex matrices such as foods.

Rapid detection of Salmonella enterica serotype Typhimurium in large volume samples using porous electrodes in a flow-through, enzyme-amplified immunoelectrochemical sensor.

Foodborne illness is a common yet preventable public health concern generating significant costs for the healthcare system, making systems to accurately detect this pathogen a topic of current research. Enzyme-based immunoassays are highly desirable because they offer shorter response times compared to traditional culture-based methods. Biosensors employing the electrochemical and optical detection of a substrate oxidized by horseradish peroxidase (HRP) have been used to successfully detect biomolecules; however, their inability to handle large sample volumes severely limits their application to food safety despite their accuracy and reliability. Here, we describe a biosensor with the capacity to process a large sample volume by utilizing an Ag/AgCl reference electrode, a platinum counter electrode, and a porous working electrode made from graphite felt coated with antibodies specific for Salmonella common structural antigens. This design allows samples to flow-through the electrode while capturing target pathogens. Following sample exposure, HRP-conjugated antibodies facilitate pathogen detection that culminates in an oxidation reaction with the output analyzed via Osteryoung square wave voltammetry. Detection limits of 1000 Salmonella enterica serotype Typhimurium cells were achieved using this newly devised flow-through, enzyme-amplified, electrochemical biosensor in samples as large as 60 mL. The low cost of the sensor allows for incorporation into disposable detection devices while its design not only broadens its applicability in sample processing but also permits the detection of various microbes by simply exchanging the antibodies.

Detection of Shiga Toxin 2 Produced by Escherichia coli in Foods Using a Novel AlphaLISA.

Amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) is comprised of a bead-based immunoassay that is used for small molecule detection. In this study, a novel AlphaLISA was developed and optimized for the detection of Shiga-toxin 2 (Stx2). Efficacy and sensitivity trials showed the AlphaLISA could detect ≥0.5 ng/mL of purified Stx2, which was comparable to the industry-standard enzyme-linked immunosorbent assay (ELISA) tests for Stx2 detection. In addition, evaluation of Shiga toxin-producing Escherichia coli (STEC)-inoculated Romaine lettuce and ground beef samples demonstrated that both the AlphaLISA and the ELISA were able to discern uninoculated samples from 1× and 10× diluted samples containing ~10 CFU/mL of STEC enriched in modified tryptic soy broth with mitomycin C for 16 h. Overall, the increased signal-to-noise ratios indicated a more robust signal was produced by the AlphaLISA compared to the ELISA and the delineation of higher toxin concentrations without the need for sample dilution implied a greater dynamic range for the AlphaLISA. Implementation of the newly developed AlphaLISA will allow for more rapid analysis for Stx2 with less manual manipulation, thus improving assay throughput and the ability to automate sample screening while maintaining detection limits of 0.5 ng/mL.

Serogroup-level resolution of the "Super-7" Shiga toxin-producing Escherichia coli using nanopore single-molecule DNA sequencing.

DNA sequencing and other DNA-based methods are now broadly used for detection and identification of bacterial foodborne pathogens. For the identification of foodborne bacterial pathogens, taxonomic assignments must be made to the species or even subspecies level. Long-read DNA sequencing provides finer taxonomic resolution than short-read sequencing. Here, we demonstrate the potential of long-read shotgun sequencing obtained from the Oxford Nanopore Technologies (ONT) MinION single-molecule sequencer, in combination with the Basic Local Alignment Search Tool (BLAST) with custom sequence databases, for foodborne pathogen identification. A library of mixed DNA from strains of the "Super-7" Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, O145, and O157[:H7]) was sequenced using the ONT MinION resulting in 44,245 long-read sequences. The ONT MinION sequences were compared to a custom database composed of the E. coli O-antigen gene clusters. A vast majority of the sequence reads were from outside of the O-antigen cluster and did not align to any sequences in the O-antigen database. However, 58 sequences (0.13% of the total sequence reads) did align to a specific Super-7 O-antigen gene cluster, with each O-antigen cluster aligning to at least four sequence reads. BLAST analysis against a custom whole-genome database revealed that 5096 (11.5%) of the MinION sequence reads aligned to one and only one sequence in the database, of which 99.6% aligned to a sequence from a "Super-7" STEC. These results demonstrate the ability of the method to resolve STEC to the serogroup level and the potential general utility of the MinION for the detection and typing of foodborne pathogens.

The Use of a Novel NanoLuc -Based Reporter Phage for the Detection of Escherichia coli O157:H7.

Rapid detection of the foodborne pathogen Escherichia coli O157:H7 is of vital importance for public health worldwide. Among detection methods, reporter phages represent unique and sensitive tools for the detection of E. coli O157:H7 from food as they are host-specific and able to differentiate live cells from dead ones. Upon infection, target bacteria become identifiable since reporter genes are expressed from the engineered phage genome. The E. coli O157:H7 bacteriophage ΦV10 was modified to express NanoLuc luciferase (Nluc) derived from the deep-sea shrimp Oplophorus gracilirostris. Once infected by the ΦV10 reporter phage, E. coli O157:H7 produces a strong bioluminescent signal upon addition of commercial luciferin (Nano-Glo(®)). Enrichment assays using E. coli O157:H7 grown in LB broth with a reporter phage concentration of 1.76 × 10(2) pfu ml(-1) are capable of detecting approximately 5 CFU in 7 hours. Comparable detection was achieved within 9 hours using 9.23 × 10(3) pfu ml(-1) of phage in selective culture enrichments of ground beef as a representative food matrix. Therefore we conclude that this NanoLuc reporter phage assay shows promise for detection of E. coli O157:H7 from food in a simple, fast and sensitive manner.

Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates.

Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 105 cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.

Accelerating sample preparation through enzyme-assisted microfiltration of Salmonella in chicken extract.

Microfiltration of chicken extracts has the potential to significantly decrease the time required to detect Salmonella, as long as the extract can be efficiently filtered and the pathogenic microorganisms kept in a viable state during this process. We present conditions that enable microfiltration by adding endopeptidase from Bacillus amyloliquefaciens to chicken extracts or chicken rinse, prior to microfiltration with fluid flow on both retentate and permeate sides of 0.2 µm cutoff polysulfone and polyethersulfone hollow fiber membranes. After treatment with this protease, the distribution of micron, submicron, and nanometer particles in chicken extracts changes so that the size of the remaining particles corresponds to 0.4-1 µm. Together with alteration of dissolved proteins, this change helps to explain how membrane fouling might be minimized because the potential foulants are significantly smaller or larger than the membrane pore size. At the same time, we found that the presence of protein protects Salmonella from protease action, thus maintaining cell viability. Concentration and recovery of 1-10 CFU Salmonella/mL from 400 mL chicken rinse is possible in less than 4 h, with the microfiltration step requiring less than 25 min at fluxes of 0.028-0.32 mL/cm(2) min. The entire procedure-from sample processing to detection by polymerase chain reaction-is completed in 8 h.

Automated immunomagnetic separation for the detection of Escherichia coli O157:H7 from spinach.

Escherichia coli O157:H7 is a major cause of foodborne illness and methods for rapid and sensitive detection of this deadly pathogen are needed to protect consumers. The use of immunomagnetic separation (IMS) for capturing and detecting foodborne pathogens has gained popularity, partially due to the introduction of automated and high throughput IMS instrumentation. Three methods for automated IMS that test different sample volumes, Kingfisher mL, Pathatrix Auto, and Pathatrix Ultra, were compared using microbiological detection of E. coli O157:H7 from buffered peptone water (BPW), in the presence of background microbial flora derived from spinach leaves, and from culture enrichments from artificially contaminated spinach leaves. The average efficiencies of capture of E. coli O157:H7 using the three methods were 32.1%, 3.7%, and 1.3%, respectively, in BPW; 43.4%, 8.8%, 2.9%, respectively, in the presence of spinach microbial flora; and 63.0%, 7.0%, and 6.3%, respectively, from artificially contaminated spinach. Despite the large differences in IMS capture efficiencies between the KingFisher and two Pathatrix methods, all three methods allowed the detection of E. coli O157:H7 from spinach that was artificially contaminated with the pathogen at relatively high (25 cfu/30 g sample) and low (1 cfu/30 g sample) levels after 4-6h of culture enrichment. The differences in capture efficiency were compensated for by the differences in sample volume used by the KingFisher mL (1 mL), Pathatrix Auto (50 mL) and Pathatrix Ultra (250 mL) instruments. Thus, despite the reduced capture efficiencies observed for the Pathatrix methods, the large increase in sample volume results in a greater number of captured cells for downstream detection resulting in improved detection sensitivity.

High-throughput biosensors for multiplexed food-borne pathogen detection.

Incidental contamination of foods by pathogenic bacteria and/or their toxins is a serious threat to public health and the global economy. The presence of food-borne pathogens and toxins must be rapidly determined at various stages of food production, processing, and distribution. Producers, processors, regulators, retailers, and public health professionals need simple and cost-effective methods to detect different species or serotypes of bacteria and associated toxins in large numbers of food samples. This review addresses the desire to replace traditional microbiological plate culture with more timely and less cumbersome rapid, biosensor-based methods. Emphasis focuses on high-throughput, multiplexed techniques that allow for simultaneous testing of numerous samples, in rapid succession, for multiple food-borne analytes (primarily pathogenic bacteria and/or toxins).

Apparent thixotropic properties of saline/glycerol drops with biotinylated antibodies on streptavidin-coated glass slides: implications for bacterial capture on antibody microarrays.

The thixotropic-like properties of saline/glycerol drops, containing biotinylated capture antibodies, on streptavidin-coated glass slides have been investigated, along with their implications for bacterial detection in a fluorescent microarray immunoassay. The thixotropic-like nature of 60:40 saline-glycerol semisolid droplets (with differing amounts of antibodies) was observed when bacteria were captured, and their presence detected using a fluorescently-labeled antibody. Semisolid, gel-like drops of biotinylated capture antibody became liquefied and moved, and then returned to semisolid state, during the normal immunoassay procedures for bacterial capture and detection. Streaking patterns were observed that indicated thixotropic-like characteristics, and this appeared to have allowed excess biotinylated capture antibody to participate in bacterial capture and detection. When developing a microarray for bacterial detection, this must be considered for optimization. For example, with the appropriate concentration of antibody (in this study, 0.125 ng/nL), spots with increased diameter at the point of contact printing (and almost no streaking) were produced, resulting in a maximal signal. With capture antibody concentrations greater than 0.125 ng/nL, the excess biotinylated capture antibody (i.e., that which was residing in the three-dimensional, semisolid droplet space above the surface) was utilized to capture more bacteria. Similarly, when the immunoassay was performed within a hydrophobic barrier (i.e., without a coverslip), brighter spots with increased signal were observed. In addition, when higher concentrations of cells (∼10(8) cells/mL) were available for capture, the importance of unbound capture antibody in the semisolid droplets became apparent because washing off the excess, unbound biotinylated capture antibody before the immunoassay was performed reduced the signal intensity by nearly 50%. This reduction in signal was not observed with lower concentrations of cells (∼10(6) cells/mL). With increased volumes of capture antibody, abnormal spots were visualized, along with decreased signal intensity, after bacterial detection, indicating that the increased droplet volume detrimentally affected the immunoassay.

An antibody microarray, in multiwell plate format, for multiplex screening of foodborne pathogenic bacteria and biomolecules.

Intoxication and infection caused by foodborne pathogens are important problems worldwide, and screening tests for multiple pathogens are needed because foods may be contaminated with multiple pathogens and/or toxic metabolites. We developed a 96-well microplate, multiplex antibody microarray method to simultaneously capture and detect Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium (S. typhimurium), as well as a biomolecule (chicken immunoglobulin G or IgG employed as a proteinaceous toxin analog) in a single sample. Microarrayed spots of capture antibodies against the targeted analytes were printed within individual wells of streptavidin-coated polystyrene 96-multiwell microtiter plates and a sandwich assay with fluorescein- or Cy3-labeled reporter antibodies was used for detection. (Printing was achieved with a conventional microarray printing robot that was operated with custom-developed microplate arraying software.) Detection of the IgG was realized from ca. 5 to 25 ng/mL, and detection of E. coli O157:H7 and S. typhimurium was realized from ca. 10(6) to 10(9) and ca. 10(7) to 10(9) cells/mL, respectively. Multiplex detection of the two bacteria and the IgG in buffer and in culture-enriched ground beef filtrate was established with a total assay (including detection) time of ca. 2.5 h. Detection of S. typhimurium was largely unaffected by high concentrations of the other bacteria and IgG as well as the ground beef filtrate, whereas a small decrease in response was observed for E. coli O157:H7. The multiwell plate, multiplex antibody microarray platform developed here demonstrates a powerful approach for high-throughput screening of large numbers of food samples for multiple pathogens and toxins.

A selective reaction of fructose bisphosphate aldolase with fluorescein isothiocyanate in chicken muscle extracts.

The present work describes the selective covalent modification of fructose bisphosphate aldolase in crude extracts of chicken breast muscle by fluorescein 5'-isothiocyanate (5'-FITC) at pH 7.0 and 35 degrees C. The modification was observed after 1 min while no other major soluble protein was labeled even after 30 min. We calculated that ca. one 5'-FITC molecule was incorporated into each aldolase tetramer after a 30 min reaction which resulted in a minimal loss of enzyme activity. The "native" structure of aldolase was required for the selective modification by 5'-FITC since high pH, high temperature, and ionic detergents either inhibited or prevented the reaction of 5'-FITC with aldolase. Certain metabolites (ATP, ADP, CTP, GTP, FBP) and erythrosin B also inhibited the 5'-FITC modification of aldolase. In contrast, F-6-P, AMP, NADH, and NAD(+) as well as free lysine and most importantly, the 6'-isomer of FITC exhibited no competition with 5'-FITC for the labeling of aldolase. Alone, the 6'-isomer of FITC did not exhibit preferential reaction when combined with aldolase. 5'-FITC-labeled and -unlabeled aldolases were not distinguished by their ability to bind to muscle myofibrils (MFs) or by their abilities to refold following reversible denaturation in urea. Structural analysis revealed that 5'-FITC-labeled a tryptic peptide corresponding to residues 112-134 in the primary structure of aldolase, a peptide that does not contain lysine, the amino acid believed to be the primary target of this reagent. Unlike chicken and rabbit muscle aldolases, chicken brain and liver aldolase isoforms along with several other aldolases derived from diverse biological sources did not exhibit this highly selective modification by 5'-FITC.

Antibody microarray detection of Escherichia coli O157:H7: Quantification, assay limitations, and capture efficiency.

A sandwich fluorescent immunoassay in a microarray format was used to capture and detect E. coli O157:H7. Here, we explored quantitative aspects, limitations, and capture efficiency of the assay. When biotinylated capture antibodies were used, the signal generated was higher (over 5-fold higher with some cell concentrations) compared to biotinylated protein G-bound capture antibodies. By adjusting the concentration of reporter antibody, a linear fluorescent response was observed from approximately 3.0 x 10(6) to approximately 9.0 x 10(7) cells/mL, and this was in agreement with the number of captured bacteria as determined by fluorescence microscopy. Capture efficiency calculations revealed that, as the number of bacteria presented for capture decreased, capture efficiency increased to near 35%. Optimization experiments, with several combinations of capture and reporter antibodies, demonstrated that the amount of bacteria available for capture (10(6) versus 10(8) cells/mL) affected the optimal combination. The findings presented here indicate that antibody microarrays, when used in sandwich assay format, may be effectively used to capture and detect E. coli O157:H7.

Comparison of enzyme-linked immunomagnetic chemiluminescence with U.S. Food and Drug Administration's Bacteriological Analytical Manual method for the detection of Escherichia coli O157:H7.

Escherichia coli O157:H7, a major foodborne pathogen, has been associated with numerous cases of foodborne illnesses. Rapid methods have been developed for the screening of this pathogen in foods in order to circumvent timely plate culture techniques. Unfortunately, many rapid methods are presumptive and do not claim to confirm the presence of E. coli O157:H7. The previously developed method, enzyme-linked immunomagnetic chemiluminescence (ELIMCL), has been improved upon to allow for fewer incidences of false positives when used to detect E. coli O157:H7 in the presence of mixed cultures. The key feature of this assay is that it combines the highly selective synergism of both anti-O157 and anti-H7 antibodies in the sandwich immunoassay format. This work presents application of a newly semi-automated version of ELIMCL to the detection of E. coli O157:H7 in pristine buffered saline yielding detection limits of approximately 1 x 10(5) to 1 x 10(6) of live cells/mL. ELIMCL was further demonstrated to detect E. coli O157:H7 inoculated into artificially contaminated ground beef at ca. 400 CFU/g after a 5 h enrichment and about 1.5 h assay time for a total detection time of about 6.5 h. Finally, ELIMCL was compared with USFDA's Bacteriological Analytical Manual method for E. coli O157:H7 in a double-blind study. Using McNemar's treatment, the two methods were determined to be statistically similar for the detection of E. coli O157:H7 in ground beef inoculated with mixed cultures of select bacteria.

Enzyme-linked immunomagnetic electrochemical detection of live Escherichia coli 0157:H7 in apple juice.

We describe the application of enzyme-linked immunomagnetic electrochemistry (ELIME) for the rapid detection of Escherichia coli O157:H7 in buffered apple juice. The ELIME technique entails sandwiching bacterial analyte between antibody-coated magnetic beads and an alkaline phosphatase-conjugated antibody. The beads (with or without bound bacteria) were localized onto the surface of magnetized graphite ink electrodes in a multiwell plate format. The enzyme substrate, 1-naphthyl phosphate, was added, and conversion of substrate to an electroactive product was measured using electrochemical detection. With this technique, detection of whole, live E. coli O157:H7 bacterial cells was achieved with a minimum detectable level of ca. 5 x 10(3) cells per ml in Tris-buffered saline or buffered apple juice in an assay time of ca. 80 min. With adjustment of pH, the ELIME response for the bacteria in either sampling medium was similar, indicating that apple juice components did not contribute to any discernible sample matrix effects.

Enzyme-linked immunomagnetic chemiluminescent detection of Escherichia coli O157:H7.

E. coli O157:H7 is a pathogenic microorganism that has been implicated in numerous cases of foodborne illnesses. A variety of rapid methods exist that show promise for the presumptive detection of this pathogen without the immediate need for incubating test samples for hours to days in microbial enrichment and culture media. In recent years, highly sensitive chemiluminescence has become a more affordable and portable detection method. Chemiluminescent detection has been coupled with the selectivity of antibodies, magnetic microparticle separation/isolation, and enzymatic signal amplification in order to develop a rapid method, termed enzyme-linked immunomagnetic chemiluminescence (ELIMCL). This work presents the application of ELIMCL to the detection of E. coli O157:H7 in pristine buffered saline with a detection limit of 7.6 x 10(3) for live cells in approx. 75 min assay time. The blocking agent casein and the surfactant Tween 20 were used to lower background luminescence and thus maximize signal-to-noise ratios. After 5.5 h of enrichment culture, ELIMCL was demonstrated to detect E. coli O157:H7 inoculated in ground beef at 10 CFU/g in a total assay time of about 7 h.