An Aptamer-Quantum Dot-Based Lateral Flow Test Strip for Cyclospora Detection

The lateral flow (LF) or immunochromatographic test strip is a staple test format in the food safety testing arsenal. However, the sensitivity of LF assays is limited partly due to antibody affinity and partly due to the visual detection limit of colloidal gold or colored latex particles. Cyclospora is an important water and foodborne parasite that requires very sensitive detection. There is a PCR-based confirmatory test available for Cyclospora. However, it requires significant sample preparation and is confined to laboratories. The PCR test cannot be used in the field or food processing plants. Therefore, Nanohmics, Inc. proposes to develop DNA aptamer (higher affinity surrogates for antibodies)-based fluorescent quantum dot (Qdot) LF test strips for Cyclospora oocysts or their extracts for rapid presumptive screening of fresh produce in the field or processing plants. Nanohmics proposes to achieve the highest possible sensitivity in a rapid (5-10 minute) field test which can be assessed by fluorescence visually with a simple handheld UV mineral light. There is a USDA SBIR Phase II-funded precedent for this technology which the proposed PI, completed and published for foodborne pathogenic bacteria in the open access journal Pathogens 3:341-355, 2014 and several other journals. In addition, Nanohmics will develop a 0.45 micron filter-based sample preparation protocol to capture and concentrate to 8-10 micron Cyclospora oocysts from fresh produce rinsates prior to LF testing. The specific Technical Objectives are:Develop, clone, and sequence new 100 base DNA aptamers against whole Cyclospora cayetanensis oocysts.Screen the new Cyclospora oocyst aptamers by ELASA (ELISA-like microplate assay) for affinity and specificity.Develop prototype aptamer-colloidal gold and aptamer-Qdot LF fluorescent test strips.Test the filter-based oocyst rinsate sample preparation, concentration and DEFT concepts for use in conjunction with the LF test strips