Non Technical Summary
Because no commercial antibodies exist against Cyclospora, Nanohmics Inc. will develop DNA aptamers to bind Cyclospora cayatanensis oocysts withhigh affinity and specificity. These aptamers will then be used to develop ultrasensitive quantum dot (Qdot)-based fluorescent lateral flow test strips for rapid (< 15 minutes) detection of Cyclospora anywhere in the food supply chain from "farm to fork" with the naked eye and a UV mineral lamp or a handheld quantitative fluorescence reader. If necessary, Cyclospora in irrigation water or rinsates from fruits and vegetables will be pre-concentrated with filters having pores of 0.45 to 5 microns in diameter and then back flushed to liberate the concentrated oocysts.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
723	4050	1110	100%

Knowledge Area
723 - Hazards to Human Health and Safety;

Subject Of Investigation
4050 - Protozoa;

Field Of Science
1110 - Parasitology;

Goals / Objectives
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

Project Methods
Technical Objective (TO)-1: Develop, clone, and sequence new 100 base DNA aptamers against whole Cyclospora cayetanensis oocysts.Nanohmics will use Bruno's extensively publishedDNA aptamer development (SELEX) method for at least 10 rounds of aptamer selection against whole Cyclospora cayetanensis oocysts. Since Cyclospora organisms are protozoan parasites, they require hosts for production and are extremely difficult to obtain. However, Dr. Bruno has already done some research and has communicated with Dr. Alexandre da Silva at the FDA (Alexandre.DaSilva@fda.hhs.gov) who graciously agreed to supply Cyclospora cayetanensis oocysts at cost s. Maximal aptamer pool affinity will be judged by the inability to heat-elute aptamers from whole cells (oocysts) in pure water at 99oC to obtain 100 bp aptamer PCR amplicon bands on electrophoresis gels. Nanohmics will clone the selected family of aptamers from the heat-elutable round into chemically competent E. coli using a Lucigen Corp. GC cloning kit. White colonies from the blue/white cloning system will be sent to Sequetech Corporation (Mountain View, CA) at a cost of $30 per DNA sequencing reaction. Nanohmics anticipates sequencing at least 100 clones. Aptamer DNA sequences will be aligned and analyzed for consensus sequences or regions of partial homology. Identical or highly homologous aptamers will be identified since such sequences often exhibit the strongest and most specific binding.TO-2: Screen the new Cyclospora oocyst aptamers by ELASA (ELISA-like microplate assay) for affinity and specificity.Nanohmics will next screen all of the unique candidate aptamer DNA sequences (≤ 100 cloned aptamer candidates anticipated) via its ELISA-like (ELASA) microplate assayto rank aptamer affinities against Cyclospora whole oocysts and lysates (detergent or 1.5M MgCl2-treated cells) according to its published protocols. The defining parameter will be absorbance at 405 nm and will be measured by a microplate reader. Nanohmics will use ten-fold serial dilutions of counted whole oocysts and lysates to determine the relative limits of detection by ELASA. Cross-reactivity will be evaluated against human red blood cells and against several species of bacteria which could interfere in the assay at the same cell concentrations.TO-3: Develop prototype aptamer-colloidal gold and aptamer-Qdot LF fluorescent test strips.The first decision to be made in the LF test strip development process is which analytical membrane and other LF components (sample pad, conjugate pad, analytical membrane and wicking pad) to use. For aptamer-based LF test strips, Bruno has already worked out the optimal choices for these components. Of some importance is thatslower analytical membranes such as Millipore's High-Flow (HF)-180 (180 seconds to wick) tend to work better for many aptamers to bind the target analytes vs. faster wicking HF-120 membranes due to greater residence time over the capture dot/line. 5'-Amine-terminated capture aptamers are used to covalently bond to the nitrocellulose on the capture line or dot using UV light to avoid the capture aptamer washing away when wicking begins. In addition, Bruno has found that a 3'-digoxigenin tag on the reporter aptamer, which is bound to streptavidin (SAv)-colloidal gold or SAv-Qdots via 5'-biotin on the other end, works well for the test line imbued with dried anti-digoxigenin monoclonal antibody. Reporter reagents are made by simply mixing the biotinylated aptamers with SAv-colloidal gold or SAv-Qdots for 30 minutes and then purifying the conjugates through a 30 kD molecular weight cut off (MWCO) spin filter to remove excess aptamers (which generally weigh < 30 kD) and eluting the capture reagent off of the top side of the spin filter in buffer.Next, matrices of all the possible capture and reporter aptamer combinations will be compared to see which aptamers pair best in the sandwich detection format. All positive (+) sandwich combinations are allowed to move forward into sensitivity (limit of detection or LOD) testing. Most preliminary testing of this type will be done with colloidal gold to keep costs down before advancing to Qdots which tend to increase the price per test, but enhance sensitivity 10-fold. Since Cyclospora oocysts are known to autofluoresce with a strong green fluorescence, Nanohmics will use red Qdots (Qdot 655) which will more easily discriminate positive tests from oocyst fluorescence background.Nanohmics will follow parallel tracts of development for the colloidal gold and Qdot 655 versions of the Cyclospora assay. Colloidal gold is very popular and inexpensive. However, ultrasensitive Qdot-based LF test strips may be required to detect the low level presence of oocysts on fresh produce surfaces. To aid in quantifying the Qdot-based strips, Nanohmics may purchase an ESEQuantTM fluorescent LF strip reader from Qiagen or may develop its own breadboard fluorescence reader in Phase II to quantify LF results. LF optimization of reagent amounts etc. and extensive cross-reactivity studies will occur in Phase II as Nanohmics prepares for FDA 510k approval and marketing of its Cyclospora test strips.TO-4: Test the filter-based oocyst rinsate sample preparation, concentration and DEFT concepts for use in conjunction with the LF test strips.Under this technical objective, Nanohmics will obtain Cyclospora cayetanensis oocysts from Dr. Alex da Silva's laboratory at the FDAand serial dilute the oocysts ten-fold down to ~ 10-100 oocysts per ml as determined by initial inoculum microscopic counts. Nanohmics will then emulate the sample preparation and pre-concentration protocol by spiking the 10-100 oocysts into 10 ml of basil/cilantro and/or berry rinsates and filtering the oocysts through a 0.45 to 5 micron pore-size Millipore filter and recounting the oocysts by microscopy. The oocysts will then be stained with DNA aptamer-biotin-SAv-red Qdot 655 conjugates and examined under the fluorescence microscope for another count using the DEFT (Direct EpiFluorescence Technique) approach. Finally, the filter membrane will be flipped, back flushed with sterile PBS buffer and the filter surface will be re-examined by fluorescence microscopy to determine how many, if any, oocysts remained on the filter after back washing.