An ultra-sensitive detection of a whole virus using dual aptamers developed by immobilization-free screening

•A pair of DNA aptamers for whole bovine viral diarrhea virus type 1(BVDV type 1) was successfully developed.•For obtaining a pair of aptamers, the advanced immobilization-free SELEX method was applied.•These two different aptamers were used in a sandwich-typed assay for the sensitive detection of BVDV type 1.•The limit of detection, 500 TCID50/ml, is comparable to the commercialized conventional real time PCR kit.

In this study, we successfully developed a ssDNA aptamer pairs by using an advanced immobilization-free SELEX method with affinity-based selection and counter-screening process at every round. By implementing this method, two different aptamers specifically binding to bovine viral diarrhea virus type 1(BVDV type 1) with high affinity were successfully screened. This aptamer pair was applied to ultrasensitive detection platform for BVDV type 1 in a sandwich manner. The ultrasensitive detection of BVDV type 1 using one of aptamers conjugated with gold nanoparticles was obtained in aptamer–aptamer sandwich type sensing format, with the limit of detection of 800 copies/ml, which is comparable to a real-time PCR method.