FOOD CONTAMINANT DETECTION AND DIRECT TO SEQUENCING

Project Summary / AbstractShigatoxin-producing Escherichia coli (STEC) is a foodborne pathogen that can cause severe illness such ashemorrhagic colitis and hemolytic-uremic syndrome (HUS) in humans. US STEC illness estimates include265,000 illnesses, 3,600 hospitalizations and numerous deaths annually. Fresh leafy vegetables have beenimplicated in many STEC foodborne disease outbreaks including a January 2018 outbreak suspected to be fromromaine lettuce. The U.S. Food and Drug Administration?s Center for Food Safety and Applied Nutrition (FDA-CFSAN) now uses Whole Genome Sequencing (WGS) and a Genome Trakr bioinformatics database as a highlyeffective regulatory tool to identify the specific source of foodborne outbreaks. One of hurdles that still needs tobe addressed however is the difficulty in isolating specific pathogens from food where selective enrichment andslow culture isolation is still required. Direct to sequencing methods to include metagenomic sequencing fordetection of STEC virulence genes directly from lettuce rinses are not possible especially for the detection ofpathogens found in low copy and as a minor proportion of the total microbial load found on foods such as lettuce.VisuGen Global is advancing a novel approach to permit the sampling, concentration, and capture of NA foundin dilute concentrations in large volumes of water or food rinses within a novel flow through NA capture chip(VisuChip). A cDNA copy of the captured NA can be made and transferred directly for sequencing. The objectiveof this phase I FDA-CFSAN SBIR is to confirm the feasibility of a using the VisuChip to capture and concentrateRNA released from microbes found in lettuce rinses and verify that VisuChip cDNA will interface withmetagenomic STEC identification. Lettuce will be spiked with a STEC strain positive for virulence genes stx1,stx2, and eae then processed in parallel with VisuChip NA capture and DNA extraction by the FDABacteriological Analytical Manual (BAM) protocol at various enrichment times. We will compare turnaround timeand limit of detection pre-enrichment and at various enrichment times following DNA 16S or metagenomicsequencing will be compared. The results of this Phase I work plan will verify the potential of VisuChip NA captureas a method to shorten STEC identification turnaround times, reduce enrichment and culture isolationrequirements, and provide feasibility for direct sample processing to sequencing for processing lettuce. Theapproach could eliminate the need to ?rst isolate and culture a contaminating STEC strain in order to detect andcharacterize it. The impact will not only be more rapid leafy green outbreak tracking to protect public health butas an approach that could improve detection of other pathogens (Salmonella, Listeria) detection and other foods(meat and poultry, cheese, etc.). Additional application of VisuGen?s sequencing platform technology in humanand veterinary, food and water disease address a greater than $100B market problem.