Food Contaminant Detection and Direct to Sequencing

Project SummaryAbstract Shigatoxin producing Escherichia coliSTECis a foodborne pathogen that can cause severe illness such as hemorrhagic colitis and hemolytic uremic syndromeHUSin humansUS STEC illness estimates includeillnesseshospitalizations and numerous deaths annuallyFresh leafy vegetables have been implicated in many STEC foodborne disease outbreaks including a Januaryoutbreak suspected to be from romaine lettuceThe U SFood and Drug Administration s Center for Food Safety and Applied NutritionFDACFSANnow uses Whole Genome SequencingWGSand a Genome Trakr bioinformatics database as a highly effective regulatory tool to identify the specific source of foodborne outbreaksOne of hurdles that still needs to be addressed however is the difficulty in isolating specific pathogens from food where selective enrichment and slow culture isolation is still requiredDirect to sequencing methods to include metagenomic sequencing for detection of STEC virulence genes directly from lettuce rinses are not possible especially for the detection of pathogens found in low copy and as a minor proportion of the total microbial load found on foods such as lettuceVisuGen Global is advancing a novel approach to permit the samplingconcentrationand capture of NA found in dilute concentrations in large volumes of water or food rinses within a novel flow through NA capture chipVisuChipA cDNA copy of the captured NA can be made and transferred directly for sequencingThe objective of this phase I FDA CFSAN SBIR is to confirm the feasibility of a using the VisuChip to capture and concentrate RNA released from microbes found in lettuce rinses and verify that VisuChip cDNA will interface with metagenomic STEC identificationLettuce will be spiked with a STEC strain positive for virulence genes stxstxand eae then processed in parallel with VisuChip NA capture and DNA extraction by the FDA Bacteriological Analytical ManualBAMprotocol at various enrichment timesWe will compare turnaround time and limit of detection pre enrichment and at various enrichment times following DNAS or metagenomic sequencing will be comparedThe results of this Phase I work plan will verify the potential of VisuChip NA capture as a method to shorten STEC identification turnaround timesreduce enrichment and culture isolation requirementsand provide feasibility for direct sample processing to sequencing for processing lettuceThe approach could eliminate the need torst isolate and culture a contaminating STEC strain in order to detect and characterize itThe impact will not only be more rapid leafy green outbreak tracking to protect public health but as an approach that could improve detection of other pathogensSalmonellaListeriadetection and other foodsmeat and poultrycheeseetcAdditional application of VisuGen s sequencing platform technology in human and veterinaryfood and water disease address a greater than $B market problem NarrativeShigatoxin producing Escherichia coliSTECis a foodborne pathogen that can cause severe illness in humans and fresh leafy vegetables have been implicated in many STEC foodborne disease outbreaks but current methods to isolate and identify the specific STEC pathogens takes days and are cumbersomeVisuGen Global LLC is developing a processing method using Nucleic AcidNAcapture direct to sequencing to enable real time STEC detection of contaminated leafy greens with rapid source identification and tracking to eliminate foodborne infection in this agriculture marketAdditional application of VisuGen s platform technology in humanveterinaryfood and water disease address a $B market problem