Screening And Application For Aptamers Of Human Noroviruses

Human noroviruses(HuNoVs)is the primary non-bacterial pathogen causing acute gastroenteritis worldwide.The detection of HuNoVs has been primarily on the molecular level.Considering the low titer of virus in the environment and food samples,it has been quite challenging to detect HuNoVs through the improved enrichment efficiency of virus particles.Screening and preparing specific antibodies are time-costing and labor-consuming.Moreover,re-systhesis,modification and preservation of antibody are difficult because of its nature of protein.Aptamer-based assay has been also applied for detection of HuNoVs through affinity binding of viral capsid,selected in a conventional systematic evolution of ligands by exponential enrichment(SELEX)process.Traditionally,screening systems are complex to operate and it places high standards for technicians and equipment.In this study,we used a novel approach to select aptamers for HuNoVs.The new approach incorporated in situ capture assay and next generation sequencing(NSG)for selecting the aptamers.P particles of HuNoV(GII.4)were purified and coated on the module to capture sequences that were specifically bound with the protein.The unbound sequences were easily removed by washing.The sequences with high affinity were amplified in the wells and selected by repeated in situ SELEX process.From the total of 30,622,226 tested sequences,two aptamers,APTL-1 and APTL-6,were selected to incorporate with in situ capture RT-qPCR assay to detect HuNoVs in samples.The sensitivity of these two aptamers was compared with porcine gastric mucin(PGM),which contains well-known viral receptors,and the reported aptamer APT-M6-2.The selected aptamer APTL-1 was comparable to PGM and slightly superior to the reported APTM6-2 aptamer for detection of HuNoVs.The results of this study lays the foundation for the enrichment for the detection of HuNoVs in the environment and food.