Generation and characterization of aptamers against grass carp reovirus infection for the development of rapid detection assay.

ABSTRACT

Grass carp reovirus (GCRV) causes devastating viral haemorrhagic disease in farmed grass carp (Ctenopharyngon idellus). As novel molecular probes, aptamers have been widely applied in rapid diagnosis and efficient therapies against virus or diseases. In this study, three single-stranded DNA (ssDNA) aptamers were selected against GCRV-infected CIK cells via SELEX (systematic evolution of ligands by exponential enrichment technology). Secondary structures predicted by MFOLD indicated that aptamers formed stem-loop structures, and GVI-11 had the lowest ΔG value of -30.84 KJ/mol. Three aptamers could specifically recognize GCRV-infected CIK cells, with calculated dissociation constants (Kd) of 220.86, 176.63 and 278.66 nM for aptamers GVI-1, GVI-7 and GVI-11, respectively, which indicated that they could serve as specific delivery system for antiviral therapies. The targets of aptamers GVI-1, GVI-7 and GVI-11 on the surface of GCRV-infected cells could be membrane proteins, which were trypsin-sensitive. Furthermore, FAM-labelled aptamer GVI-7 could be applied to detect GCRV infection in vivo. It is the first time to generate and characterize aptamers against GCRV-infected cells. These aptamers have great potentials in development of rapid diagnosis technology and antiviral agents against GCRV infection in aquaculture.