Performing Department
BIOSYSTEMS AG EGR
Non Technical Summary
According to a USDA Economic Research Service report in 2018, the economic burden due to 15 foodborne pathogens was estimated close to $18 billion in the U.S. Salmonella and Campylobacter caused the most reported bacterial foodborne illnesses. Epidemiological studies have suggested that products of poultry origin are among the most common vehicles for transmission of Salmonella spp. and Campylobacter spp.. In the U.S., poultry meat consumption is increasing per capita per year compared to the rest of the world. Bacteria from farm production, slaughterhouses, and processing equipment contaminate carcasses and processed meat products. The presence of very low levels of live bacteria in food samples can render the food hazardous. Microbiological culture-based methods are limited in their ability to provide timely data. Currently available rapid detection systems are often laborious, expensive, and require laboratory facilities. They also lack the ability to quickly enrich and detect targets in large samples. There is an unmet need for rapid, low-cost, onsite, and accurate detection of foodborne agents in large samples. Thus, the goal of this proposal is to rapidly monitor the temporal dynamics of Salmonella and Campylobacter infection in poultry farms and slaughter facilities using a nanoparticle-based biosensor with capabilities for rapid and simultaneous enrichment, detection, and reporting of multiple agents.This research is a partnership between Michigan State University and Tuskegee University for food safety. Due to its simplicity and affordability, the cellphone-enabled SMART biosensor can be used onsite to monitor and generate internal information on the effectiveness of antimicrobial intervention strategies, provide temporal dynamics of the infection rate at control points, and provide early information on potential development of antimicrobial resistance of the bacteria to the treatment and prevention strategies.Although the biosensor may not be able to replace culture and may not be as sensitive as real-time polymerase chain reaction (RT-PCR), it will allow high frequency testing and support faster decision-making by producers and operators, resulting in the implementation of pathogen reduction programs that will prevent these pathogens from entering the food supply chain, thus protecting public health, reducing foodborne illness outbreaks, and sustaining the availability of nutritious foods. The biosensing technology will also be able to guide producers identify specific sites in their facilities that need improved sanitation and intervention strategies, resulting in higher efficiency of operation, increased productivity, and overall higher business profitability.
Animal Health Component
0%
Research Effort Categories
Basic
20%
Applied
40%
Developmental
40%
Goals / Objectives
Poultry is one of the most consumed animal-based protein in the U.S. Unfortunately, poultry and poultry products are considered as major vehicles of Salmonella and Campylobacter infection in humans. Salmonella and Campylobacter are the top two leading causes of bacterial foodborne diarrheal illnesses in the U.S. causing close to a million cases of illnesses by each organism every year. According to a USDA Economic Research Service report in 2018, the economic burden was $4 billion due to Salmonella (non-typhoidal) infection and $2 billion due to Campylobacter infection. Currently, there is a technology gap for the simultaneous extraction, concentration, purification, and detection of multiple pathogens in field settings, close to the contamination site. Thus, the goal of this proposal is to rapidly monitor the temporal dynamics of Salmonella and Campylobacter infection in poultry farms and slaughter facilities using a nanoparticle-based biosensor with capabilities for rapid and simultaneous enrichment, detection, and reporting of multiple agents. The specific objectives are: (1) optimize the Site-enriched-Multi-Array-Reporting Biosensing Technology (SMART) for rapid detection of Salmonella spp. and Campylobacter spp. in large samples; (2) optimize a cellphone-based data capture, analysis, and reporting of the biosensor signal output; and (3) validate the cellphone-enabled SMART biosensor for simultaneous detection of Salmonella spp. and Campylobacter spp. in selected poultry farms and slaughter facilities.
Project Methods
The specific objectives of this project are: (1) optimize the Site-enriched-Multi-Array-Reporting Biosensing Technology (SMART) for rapid detection of Salmonella spp. and Campylobacter spp. in large samples; (2) optimize a cellphone-based data capture, analysis, and reporting of the biosensor signal output; and (3) validate the cellphone-enabled SMART biosensor for simultaneous detection of Salmonella spp. and Campylobacter spp. in selected poultry farms and slaughter facilities.The SMART biosensor combines microbial enrichment, biosensing assay, and smartphone-based data analytics designed to empower proactive and effective decision making in food safety. The microbial enrichment step is facilitated by the glycan-coated magnetic nanoparticles (GMNPs); the diagnostic test is facilitated by the thiolated gold nanoparticles (GNPs) for the detection of multiple target agents and genes. GMNPs are used to enrich the microbial contaminants in the sample; GNPs are used to detect and confirm the presence of multiple pathogens and multiple target genes in the sample. The solution changes color after adding a signal buffer: red for the presence of the target DNA due to the formation of bacteria-specific complex; blue if the target DNA is absent. The biosensor can be conducted at higher frequency due to its simplicity, speed, and affordability. Although GMNPs lack the specificity to the pathogens, they have shown the capacity to extract and concentrate multiple bacteria, increasing the sensitivity of the DNA-based biosensing assay.Target genes for Salmonella spp. and Campylobacter spp. will be identified. The invA gene has been frequently used in biomolecular detection of Salmonella spp.. Additional genes will be explored, such as hilA, fljB, fliC, bcfD, phoP, siiA, and others as necessary. Specific Campylobacter genes, such as omp50, mapA, ceuE, and flaA, could be used for specific detection of C.jejuni and C. coli. Briefly, sequences of these genes will be retrieved from the NCBI microbial genome-sequencing database. Unique targets will be identified after thorough study of multiple alignment of these sequences. Once target sequences are identified, a BLAST search directly from NCBI and PATRIC databases will be performed to verify the uniqueness of the target sequence. The target DNA will further be validated for their specificity. We will use multiple target genes and multiple oligonucleotide probes of various lengths in the SMART biosensor.For objective 1, the following studies will be conducted to optimize the enrichment and biosensing assays under lab conditions:Optimize sampling protocols for collecting manure samples from poultry farms and scald-water samples from slaughterhouses.Optimize sample preparation that is compatible with enrichment and biosensing assaysConduct multi-Array extraction and enrichment within 10-20 minDetermine the concentration factor of the enrichment assayConduct genomic DNA extractionConduct multi-array DNA detection within 30-40 minDetermine the analytical sensitivity of the biosensorDetermine the analytical specificity of the biosensorDetermine the analytical precision of the biosensorDetermine the analytical accuracy of the biosensorValidate the biosensor performance using PCRFor objective 2, the following studies will be conducted to optimize the smartphone-based data capture and analysis:Design and code the algorithm for data capture using the smartphone cameraDesign and code machine learning algorithm for data analysisDesign and code the App for analytical output displayDesign and code the App for user-friendly operation and human-machine interfaceDesign and code the App for display of temporal dynamics of infectionFor objective 3, the following studies will be conducted to assess the performance of the enrichment and biosensing assays in farm and field samples:Optimize sample collection from farm and slaughterhouse facilitiesCollect fecal and water samples from various locations in farms and slaughterhousesConduct multi-array enrichment on-farmConduct genomic DNA extraction on-farmConduct multi-array DNA detection on-farmCompare biosensor results with PCRMonitor the temporal dynamics of infection due to Salmonella and CampylobacterActivities cutting across the three objectives -System development to assess technology readiness and adoption by the poultry industry:Assess compatibility of enrichment and biosensing assays with industry practice to ensure smooth adoption of technologies into the farm and slaughterhouse operationMeet with collaborators and other stakeholders to assess technology readinessAssess the benefits, costs, competitors, constraints by potential users, and other parameters to assess the readiness of the technology for on-farm useBenchmark other existing tests, such as culture and PCR, with biosensor technologyMeet with representatives of industry associations, poultry extension specialists, and other stakeholders to evaluate the significance of the findings, and how that information will impact decision making at specific sites in the field