P3-47 The Influence of Four Food Matrices on Aptamer Enrichment Targeting the P-Domain of Norovirus

Wednesday, August 3, 2016
America's Center - St. Louis
Katja Schilling, U.S. Food and Drug Administration-Gulf Coast Seafood Laboratory, ORISE, Dauphin Island, AL
Jacquelina Woods, U.S. Food and Drug Administration-Gulf Coast Seafood Laboratory, Dauphin Island, AL
Introduction: Norovirus is the leading cause of gastroenteritis and foodborne illness in the US. Low virus numbers and the inability to culture norovirus pose a challenge for norovirus detection in contaminated foods. Using aptamers as extraction tools could greatly benefit virus isolation from food, enabling the development of custom extraction procedures.

Purpose: The purpose of this study is to investigate the influence of food matrices on aptamer enrichment, to assess if food matrices present during the target binding step of SELEX will alter aptamer enrichment.

Methods: Sixteen rounds of SELEX were completed in five parallel approaches: four with food matrices (strawberries, lettuce, whole oyster, and oyster diverticulum) and one in selection buffer. After target binding of the sequence pools from the final rounds was confirmed, the sequence pools were cloned, and fifty sequences from each approach examined for similar motifs and identical sequences. Binding characteristics of the sequence pools and candidates, in the presence of each food matrix and salmon sperm DNA, were determined via filter retention assays in combination with autoradiography, using α-thrombin and TBA1 as experiment control.

Results: The sequence pools of each SELEX approach exhibited different affinities to the P-Domain, with the buffer approach exhibiting the highest and the oyster diverticula approach the lowest target binding. The 50 sequences from the final rounds of the lettuce and buffer approaches each contained 8 identical sequences, whereas the sequence pools from both oyster approaches contained 41 to 46 random sequences. The two candidates which showed specific target binding (KDs: 30 nM and 80 nM), even in presence of food matrix, were enriched in the buffer approach.

Significance: This novel investigation of the food matrix influence on aptamer enrichment and the assessment of aptamer target binding in the presence of foods helps evaluate the utility of aptamers for food analysis.