Fluorescent Aptamer Test Strips for Food Safety Testing

The lateral flow (LF) or immunochromatographic (IC) 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. Operational Technologies Corporation (OpTech) has developed hundreds of high affinity candidate DNA aptamers against foodborne pathogenic bacterial surface markers such as E. coli intimins (eae gene products), O157 LPS, Campylobacter and Salmonella surface proteins, Listeria flagellin proteins and Listeriolysin O (LLO) many of which it has patented, published and now sells on its website (www.OTCBiotech.com). In Phase 1, OpTech developed and screened longer (200 base) "multivalent" DNA aptamers containing several of its highest affinity aptamer sequences by ELISA-like (ELASA) microplate assay. These lengthier aptamers appear to have enhanced avidity and specificity versus the older 72 base aptamers in preliminary ELASA screening. OpTech developed the new 200 base aptamers with USDA SBIR funding against the "Big 6" non-O157 Shiga-toxin producing E. coli (STEC) serogroups of lipopolysaccharides (LPS; O26, O45, O103, O111, O121, and O145) and has begun to incorporate these into LF strips having multiple detection lines per strip to help satisfy the new USDA requirement for "Big 6" non-O157 STEC detection.As a preliminary demonstration of how aptamer LF test strips could work, during Phase 1, OpTech screened a number of general botulinum toxin, Campylobacter, E. coli, Listeria, and Salmonella aptamer sandwich pairs in LF test strip formats using both colloidal gold and red quantum dots (Qdot 655). Promising aptamer pairs were identified for most of these analytes and the best studied E. coli system produced a limit of detection (LOD) of about 3,000 cells/test by colloidal gold and < 300 cells by Qdot 655-aptamer-based LF visual tests under a UV light. This work was published by Bruno in the open access journal Pathogens 3:341-355, 2014. Due to this clear enhancement of sensitivity via the use of red Qdots and aptamers, OpTech wishes to fully develop a whole line of fluorescent Qdot-aptamer foodborne pathogen LF test strips for commercialization via direct sales and/or product licensing to larger companies. OpTech is currently working with a food safety industry consultant (www.XgeneX.com) to develop partnering relationships and/or licensing deals.The broader impacts of this SBIR project will include enhanced food safety for the public as well as better on-site decision making ability for food processing and packing facilities to decide if foods can be sold or should be discarded. In addition, aptamer-based fluorescent LF test strips can be developed to detect a broad array of clinical, veterinary, environmental pollutant or other analytes. Initially, food processors will be OpTech's main customers. However, the customer base could expand dramatically as OpTech moves fluorescent aptamer-LF test strip technology into the homeland security and medical arenas to detect chemical and biological terrorist threat agents, infectious diseases in hospitals, patient wounds or contaminated raw materials in pharmaceutical production facilities, etc.