Abstract
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.
Highlights
Human noroviruses (HuNoV) are the most common cause of acute viral gastroenteritis worldwide [1]
While molecular amplification methods are commonly used in public health settings, these methods are rarely used in clinical diagnostic laboratories in the U.S Detection of HuNoV in food and environmental samples is even more complicated because virus concentrations are so low in these samples that it is necessary to perform labor intensive and relatively inefficient pre-concentration step(s) prior to detection [8]
Snow Mountain virus (SMV), the prototype genogroup II, genotype 2 (GII.2) HuNoV and the target for aptamer selection, and Norwalk (NV) the prototype genogroup I, genotype 1 (GI.1) strain were obtained as stool specimens originating from a human challenge study
Summary
Human noroviruses (HuNoV) are the most common cause of acute viral gastroenteritis worldwide [1]. High virus concentrations in the feces and vomitus of infected individuals, lengthy environmental persistence, and resistance to many commonly used sanitizers and disinfectants all contribute to the high degree of transmissibility of HuNoV [5]. Despite their public health significance, routine detection of HuNoV in community settings or in food and environmental samples, is limited. While molecular amplification methods ( reverse transcriptase quantitative PCR or RT-qPCR) are commonly used in public health settings, these methods are rarely used in clinical diagnostic laboratories in the U.S Detection of HuNoV in food and environmental samples is even more complicated because virus concentrations are so low in these samples that it is necessary to perform labor intensive and relatively inefficient pre-concentration step(s) prior to detection [8]
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