Key result
Single-stranded DNA aptamer binds multiple Human Norovirus strains, detecting ~10 RNA copies in contaminated lettuce.
ssDNA aptamers, particularly aptamer 25, demonstrate broad reactivity and potential utility as diagnostic reagents for the capture and detection of human norovirus in clinical and food samples.
Aptamer 25 may aid norovirus detection in food matrices; leaves open clinical or regulatory adoption without further validation.
Human noroviruses (HuNoV) are the leading cause of acute viral gastroenteritis and an important cause of foodborne disease. Despite their public health significance, routine detection of HuNoV in community settings, or food and environmental samples, is limited, and there is a need to develop alternative HuNoV diagnostic reagents to complement existing ones. The purpose of this study was to select and characterize single-stranded (ss)DNA aptamers with binding affinity to HuNoV. The utility of these aptamers was demonstrated in their use for capture and detection of HuNoV in outbreak-derived fecal samples and a representative food matrix. SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used to isolate ssDNA aptamer sequences with broad reactivity to the prototype GII.2 HuNoV strain, Snow Mountain Virus (SMV). Four aptamer candidates (designated 19, 21, 25 and 26) were identified and screened for binding affinity to 14 different virus-like particles (VLPs) corresponding to various GI and GII HuNoV strains using an Enzyme-Linked Aptamer Sorbant Assay (ELASA). Collectively, aptamers 21 and 25 showed affinity to 13 of the 14 VLPs tested, with strongest binding to GII.2 (SMV) and GII.4 VLPs. Aptamer 25 was chosen for further study. Its binding affinity to SMV-VLPs was equivalent to that of a commercial antibody within a range of 1 to 5 µg/ml. Aptamer 25 also showed binding to representative HuNoV strains present in stool specimens obtained from naturally infected individuals. Lastly, an aptamer magnetic capture (AMC) method using aptamer 25 coupled with RT-qPCR was developed for recovery and detection of HuNoV in artificially contaminated lettuce. The capture efficiency of the AMC was 2.5-36% with an assay detection limit of 10 RNA copies per lettuce sample. These ssDNA aptamer candidates show promise as broadly reactive reagents for use in HuNoV capture and detection assays in various sample types.
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Escudero‐Abarca et al. (2014) studied Human Norovirus. ssDNA aptamers (aptamer 25) vs. Commercial antibody and negative controls was evaluated on Binding affinity and capture efficiency for Human Norovirus strains. Single-stranded DNA aptamer 25 demonstrated broad binding affinity to multiple Human Norovirus strains and achieved a capture efficiency of 2.5–36% with a detection limit of 10 RNA copies in artificially contaminated lettuce.
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