Capture and Detection of a Representative Human Norovirus Strain Using Target-specific Nucleic Acid Aptamers: Proof of Concept

Introduction: Human noroviruses (NoV) are the most common cause of acute viral gastroenteritis and a leading cause of foodborne disease.  Detection is an important tool in our attempt to reduce disease burden. Aptamers [small, single-stranded (ss) DNA or RNA molecules with target binding affinity] are emerging ligands to facilitate pathogen capture and detection. They demonstrate advantages over traditional capture ligands like antibodies, including reduced cost, ease of production, and high stability.

Purpose: To develop capture and detection methods using pre-selected ssDNA aptamers with binding specificity to Snow Mountain virus (SMV), a prototype human NoV strain.

Methods: Two assay designs were produced.  In the aptamer magnetic capture (AMC) design, aptamers (NVII-13, 24) were used as capture ligands, with subsequent detection using virus-specific RT-qPCR.  In the second assay (sandwich-based), SMV was captured using antibody-bound beads, which were then exposed to a SMV-specific aptamer (NVII-22).  Detection was achieved by amplification of the aptamer by qPCR.

Results: The mean capture efficiency (%) of aptamers NVII-13 and NVII-24 using the AMC-RT-qPCR method as applied to serially diluted SMV stock ranged from 2.5 - 42%, and increased (improved) as virus titer decreased (from 4 log – 1 log GEC/ml). The lower limit of detection (LoD) of the assay was 1 log genome equivalent copies (GEC) SMV/ml.  For the sandwich assay format, sample positivity was established as Ct values > 2-3 times the standard deviation associated with the negative control.  This criterion was met for samples containing between 1 and 4 log GEC SMV/ml, equating to a lower limit of detection of 1 log GEC/ml.  Both of the aptamer-based assays had a 3 log lower (better) LoD when compared to similar immunomagnetic separation- RT-qPCR detection assays.

Significance: This study shows proof-of-concept that nucleic acid aptamers can be used for capture and detection of human NoV, with excellent detection limits.  Further studies are underway to evaluate their usefulness in NoV detection in foods.