Inverse PCR

Inverse PCR was used to confirm the presence of the reporter gene replicon inside the PsVs. A PCR amplification-product will only be produced in the presence of a recircularised replicon (Fig. 3). DNA was extracted from the fractions in either the presence or absence of proteinase K (protK) for PsV capsid digestion. Fractions were mixed with PBS in a 2:3 ratio with protK or not. The samples were incubated at 55 °C for 3 h followed protK inactivation at 90 °C. The samples where cleaned using QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions. Primers Rep_For (5′-TCCATCGTGCGTCAGATTTGCG-3′) and SEAP_Q (5′-GGCTCTGTCCAAGACATACAATGTA-3′) were used for replicon amplification. The presence of 2.1 or a 2.3 kb product confirms the presence of either the mSEAP or iSEAP replicons, respectively.