Quantification of Virion-Sense and Complementary-Sense DNA Strands of Circular Single-Stranded DNA Viruses

Circular ssDNA viruses are ubiquitous and can be found in both prokaryotes and eukaryotes. To understand the interaction of ssDNA viruses with their hosts, it is important to characterize the dynamics of viral sense (VS) and complementary-sense (CS) viral strands during the infection process. Here, we present a simple and rapid protocol that allows sensitive and accurate determination of the VS and CS strands generated during viral infection.The method consists of a two-step qPCR in which the first step uses a strand-specific (CS or VS) labeled primer and T4 DNA polymerase that lacks strand displacement activity and makes a single copy per VS or CS strand. Next, the T4 DNA polymerase and unincorporated oligonucleotides are removed by a silica membrane spin column. Finally, the purified VS or CS strands are quantified by qPCR in a second step in which amplification uses a tag primer and a specific primer. Absolute quantification of VS and CS strands is obtained by extrapolating the Cq data to a standard curve of ssDNA, which can be generated by phagemid expression. Quantification of VS and CS strands of two geminiviruses in infections of Solanum lycopersicum (tomato) and Nicotiana benthamiana plants using this method is shown.