Rolling-circle amplification for the detection of active porcine circovirus type 2 DNA replication in vitro

Porcine circovirus type 2 (PCV2) infections in pigs have diverse clinical presentations and are considered economically important diseases worldwide. However, despite intensive research, the early pathogenesis of PCV2 and the primary target cells for PCV2 infections and replication are still unknown. Rolling-circle amplification (RCA) is an amplification technique for small, circular DNA templates that essentially mimics rolling-circle replication in vitro. In this study, the amplification of PCV2-specific DNAs using randomly primed RCA has been demonstrated. This novel approach has circumvented the normal requirement for conventional virus isolation procedures for the characterization of PCV2 DNAs from clinical samples. In addition, the potential utility of a strand-specific derivative of RCA was further investigated. Specifically, strand-specific RCA for the detection of active virus replication following the amplification of complementary sense PCV2 DNAs, which occur as double-stranded replicative intermediates that are present only during de novo viral DNA replication both in vitro and in vivo has been demonstrated.