Period of performance:
Bacterial contamination and infection is a significant problem to public health, food, industry, environmental biosafety, and many other areas. However, current methods for detecting bacteria in medical, veterinary, agricultural, food processing, industrial and other contexts are slow, require specialized personnel or equipment to execute, and are often expensive. These drawbacks result from the need to overcome the following challenges: 1) separating bacteria from the sample matrix to prevent interference from the matrix and competing non-pathogenic bacteria present in the sample, 2) achieving sufficient signal from the analyte over background noise, and 3) discriminating live from dead bacteria. In order to overcome these hurdles, current methods for bacterial pathogen detection in the food industry rely on an enrichment step (or steps) to increase the number of target pathogen cells over the background microbial flora. Thus, current processes require a large time lag between sampling and final readout, during which time the sampled conditions may have changed and the results of the assay cannot be confidently utilized to act on contamination in already widely distributed food products. As a result, significant economic costs are incurred by the food industry due to frequent and expensive recalls and public health is affected by unpredictable outbreaks in the food supply. There is a large unmet need for technologies that can provide quick, sensitive, and specific detection of foodborne pathogens to enable proactive, convenient, and rapid food safety programs that reduce costs and threats to human health.
Our research aim is to develop a technical solution that enables near-real-time, on-site, specific, sensitive, easy-to-use, and point-of-care diagnostics for microbial pathogens, which can be directly implemented in food-related industries. We propose to combine a powerful microfluidics-enabled concentration and separation platform for sample preparation that leads into a downstream detection step using pathogen-specific bacteriophages that are engineered to provide highly sensitive and specific detection of target bacteria. Our microfluidic sample preparation platform concentrates bacteria and separates them from the sample matrix, thus minimizing matrix interference, removing the need for high sample dilution, and making it even easier for the downstream bacteriophage-based assay to achieve microbial detection. Our phages have natural specificity towards target microbial pathogens and can readily distinguish between pathogens and other microbial flora. The phages are genetically engineered to strongly express a highly active form of luciferase once they have infected a target pathogen, which produces photons that are easily detected with a luminometer as a marker of pathogen presence. Our integrated platform is highly flexible, supported by strong preliminary data, and can be optimized for a wide range of foods (e.g., milk, meat, vegetables) that pose a broad spectrum of technical challenges. In summary, our transformative technologies will result in pathogen diagnostic assays that require minimal enrichment, thus speeding the time-to-detection, and that are easy to use and do not require technically sophisticated operators to run, thus facilitating widespread adoption in the food safety industry and eventually other areas of high importance (water, clinical, oil and gas, etc.).