Development and Characterization of Nucleic Acid Aptamers for the Detection of Human Norovirus across a Broad Group of Genotypes

Introduction: Human noroviruses (HuNoV) are a leading cause of foodborne illness. There is no easy way to detect HuNoV contamination in food and environmental samples. A major reason for this is the need to concentrate and purify small numbers of viruses from the sample matrix prior to detection using reverse transcription (RT)-qPCR. Aptamers [small, single-stranded (ss)DNA or RNA molecules that naturally fold into complex three-dimensional shapes] are emerging ligands for pathogen capture. They demonstrate advantages over traditional capture ligands like antibodies, including reduced cost, ease of production and modification, and improved stability. There is also evidence that aptamers may have broader strain reactivity than antibodies.

Purpose: To create and characterize ssDNA aptamers with binding specificity to the P domain (putative capsid binding domain) of a prototype HuNoV strain.  

Methods: The P protein of a HuNoV GII.4 2006b strain was used as target for selection of ssDNA aptamers. Promising candidates were characterized for binding affinity and specificity using an Enzyme-Linked Aptamer Sorbant Assay (ELASA) applied to a panel of 14 different HuNoV virus-like particles (VLPs). VLP binding was confirmed with stool samples derived from infected individuals.  

Results: Two promising aptamer candidates--M1 and M6-2--containing low DG (-12.51 and -13.75, respectively) and unique secondary structures were selected for characterization. Both demonstrated strong binding affinity to GI.7, GII.2, GII.4, and GII.7 VLPs; and M6-2 showed moderate to high binding to all but one remaining VLP. Both aptamers also showed statistically significant binding (P < 0.001) when applied to HuNoV-positive stool samples obtained from outbreaks.

Significance: This is the first report of ssDNA aptamer selection and specificity characterization using easily-expressed HuNoV purified capsid protein. The resulting aptamers had relatively broad reactivity and performed well in clinical stool samples. They are currently being evaluated for use in capture and detection of HuNoV in foods.