Selection, Characterization and Application of Nucleic Acid Aptamers for the Capture and Detection of Human Norovirus Strains

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.

Human Norovirus Aptamer Exhibits High Degree of Target Conformation-Dependent Binding Similar to That of Receptors and Discriminates Particle Functionality

“…The docking simulation predictions were confirmed by mutational analysis of aptamer M6-2. Native M6-2 exhibited binding, considered a VLP/no-VLP ratio of >2.0 per convention (8, 19, 21), for all of the aptamer concentrations tested, except at 0.001 µM. None of the other mutated aptamers displayed binding at any concentration, confirming docking simulation predictions.…”

“…All three binding assays (HBGA, antibody, and aptamer) were derived from one another (21, 30). All three also utilized horseradish peroxidase (HRP) as a reporter and were developed with the same substrate system.…”

“…The assays and analyses used are easily adaptable to other targets, as aptamers have been successfully used to detect infectious virus in stool samples (19, 21) with minimal modification. The most recent strain of the pandemic human norovirus genotype that causes the majority of human norovirus cases (GII.4 Sydney) was chosen as the model for this proof-of-concept study.…”

Gravimetric Viral Diagnostics: QCM Based Biosensors for Early Detection of Viruses

“…Aptamers have already been produced for a wide variety of viruses. A few prominent examples are HIV [129,130], HepBV [131], EBOV [132], severe acute respiratory syndrome (SARS) [133,134], norovirus [135], rabies virus [136], vaccinia virus [137,138], dengue virus [128], and influenza viruses [139,140].…”

Miniaturized electrochemical sensor modified with aptamers for rapid norovirus detection

“…Parameters of SWV experiment were chosen as step potential of 4 mV, amplitude of 5 mV and frequency of 10 Hz. The ferricyanide/ruthenium hexamine reporter system is able to enhance the electrochemical signal [5,6] ) in the precedent step, thereby amplifying the electrochemical readout. The magnitude of current signal is correlated with the amount of accessible aptamers.…”