Project SummaryThe Food and Drug Administration (FDA) regulates pet food, requiring it is safe to eat, containsno harmful substances, is produced using sanitary conditions, and is truthfully labeled.However, the foods do not need pre-market approval because they are made with ingredientsconsidered safe. Pet food adulteration has been shown to occur in plants during processing,resulting in illness in animals and humans related to foodborne pathogens. Plant processingmay also result in cross-contamination between products used for pet food production. This canresult in pet foods having unintended ingredients, leading to false labelling. Whether intentionalor not, adulteration of pet food with DNA from unlabeled genera has been previously detected.Current methods used to detect foodborne pathogens are labor-intensive and may take severaldays to obtain actionable results. Additionally, samples likely have to been sent to severaldifferent labs for comprehensive testing (e.g. for virus, bacteria, and parasites) and for detectionof the genus of the meat source in the food. Additional testing capabilities, includingcomprehensive methods for rapid detection and initial characterization, would be beneficial forrapid recall of adulterated food.We propose to develop a targeted next-generation sequencing (NGS) panel, which incorporatesprimers for multiple pathogens associated with foodborne illness, for detection and initialcharacterization of the involved pathogen(s), and for characterization of the meat included in theproduct. Primers can be incorporated into the NGS workflow for multiplex PCR prior tosequencing. Use of primers allows for standardization of the NGS protocol for consistentperformance across multiple labs and eases the use and implementation of the technology bythose already familiar with PCR and Sanger sequencing. This renders implementation of NGSrelatively simple in the diagnostic lab setting, even though NGS bioinformatics pipelines stillneed further development and validation for standardization across labs. The simplistic analysisof the data generated from a targeted NGS panel mitigates this limitation. Additionally, as aresult of the initial PCR amplification steps, the test can be performed directly from the clinicalsample or following an enrichment process. All of these advantages suggest that targeted NGScan be used to provide a comprehensive diagnostic assay for the detection of foodbornepathogens with initial pathogen characterization and detection of meat source genera, that hasa more rapid turn-around time than currently used methods.