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Application of Droplet Digital Polymerase Chain Reaction (PCR) to Improve Food Safety
Joseph Capobianco, PhD
Research Materials Engineer
United States Department of Agriculture
Foodborne illness is a common, underreported, costly and yet preventable public health problem that contributes significantly to the cost of health care. The consequences of consuming contaminated food are extensive, with an estimated 47.8 million episodes of illness, 127,839 hospitalizations, and 3,037 deaths in the U.S. alone each year. As a result producers and regulatory agencies invest time, money, and resources into testing foods for the presence of pathogen contamination. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens because they are generally time-efficient, sensitive, specific and labor-saving.
A major challenge in food safety is to identify traits that can selectively identify bacteria that are harmful to human health, such as Shiga toxin production Escherichia coli (STEC), from those that are not and simply exist in/on the foods we eat. Many of the current accredited methods for the detection of STEC in foods rely on a PCR-based screen for the pathotype-specific genetic markers stx and eae. Unfortunately, these methods often suffer from a high rate of false-positive results, partly due to the inability of the methods to determine if both stx and eae originated from a single organism as opposed to a mixture of organisms. This research was undertaken to evaluate a droplet digital PCR (ddPCR)-based method that could reduce the false positive rate associated with current STEC screening methods by confirming that both stx and eae reside within the same cell. The ddPCR system used in this study, dd-Check STEC Solution (Bio-Rad), works by partitioning intact cells into emulsion droplets, which subsequently undergo multiplexed endpoint PCR. This enables the assay to differentiate between samples where a single organism contains both stx and eae from samples in which stx and eae reside in different organisms. Comparison were made between the dd-Check STEC Solution and two commercially available real-time PCR assays using over 35 unique simulations of STEC contamination in ground beef with the dd-Check STEC Solution demonstrating an equivalent sensitivity to the established PCR screening techniques.
Moreover, the results indicated that the dd-Check STEC Solution has the potential to reduce the number of false-positive results when screening for STEC because the assay could distinguish between the co-existence of multiple genes within the same cell from a mixed microbial population; specifically, the stx and eae genes. Ultimately, this system will result in cost-savings by reducing the man-hours and testing expenses associated with the evaluation of false-positive samples. Furthermore, this would enable more samples to be analyzed, which could reduce the probability of contaminated foods reaching consumers.
Joseph Capobianco, PhD, is a research Materials Engineer at the United States Department of Agriculture and a member of the Molecular Characterization of Foodborne Pathogens Research Unit. This team is actively engaged in research focused on addressing current unmet needs in food safety; specifically developing novel processes for rapid and cost effective sample preparation along with field portable detection methods for identifying foodborne pathogens. The research is directed toward implementation of on-line or near real-time detection and/or typing technologies to be used by regulatory agencies and the food industry to prevent contaminated food from reaching the marketplace.
Dr. Capobianco graduated summa cum laude and number one of his class from Drexel University with a BS degree in Biomedical Engineering while working a full-time job as a sheet metal mechanic. Subsequently, he obtained a PhD in Materials Engineering under the guidance of Wei-Heng and Wan Y. Shih. As an entrepreneur, Dr. Capobianco founded two companies, ACI Materials and TBT Group Inc., and he actively serves as an executive officer and board member. He also served as director of research and development at ESL ElectroScience, prior to the acquisition by Ferro Corporation.