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Six hundred million cases of disease and roughly 420,000 deaths occur globally each year due to foodborne pathogens. Current methods to screen and identify pathogens in swine, poultry, and cattle products include immuno-based techniques (e.g., immunoassay integrated biosensors), molecular methods (e.g., DNA hybridization and PCR assays), and traditional culturing. These methods are often used in tandem to screen, quantify, and characterize samples, prolonging real-time comprehensive analysis. Next-generation sequencing (NGS) is a relatively new technology that combines DNA-sequencing chemistry and bioinformatics to generate and analyze large amounts of short- or long-read DNA sequences and whole genomes. The goal of this project was to evaluate the quantitative capabilities of the real-time NGS Oxford Nanopore Technologies’ MinION sequencer through a shotgun-based sequencing approach. This investigation explored the correlation between known amounts of the analyte (lambda DNA as a pathogenic bacterial surrogate) with data output, in both the presence and absence of a background matrix (Bos taurus DNA). A positive linear correlation was observed between the concentration of analyte and the amount of data produced, number of bases sequenced, and number of reads generated in both the presence and absence of a background matrix. In the presence of bovine DNA, the sequenced data were successfully mapped to the NCBI lambda reference genome. Furthermore, the workflow from pre-extracted DNA to target identification took less than 3 h, demonstrating the potential of long-read sequencing in food safety as a rapid method for screening, identification, and quantification.

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.

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

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.

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.

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.

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 (~108 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 (~106 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.