Viral Genome Extraction and Quantification

Due to the limited sample volume and the decision to use 1 mL for the passaging of samples in cell culture, 10 µl of each sample was diluted in 90 µl of the respective medium (PBS, ZB, or FCS) to receive 100 µl for the extraction of DNA. Nucleic acid extraction was done using the NucleoMag Vet kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions on the KingFisher Flex System (Thermo Scientific, Darmstadt, Germany), with some modifications in the volume of certain substances [22 (link)]. For the four CaPV isolates, the viral genome loads were determined using a pan-capripox real-time qPCR amplifying a region in the p-32 gene [2 (link)] in combination with a modified probe [23 (link)]. PCPV genomes were detected using a real-time qPCR assay targeting the B2L gene [24 (link)]. An internal control DNA (IC2-DNA) was added for the control of successful DNA extraction [25 (link)]. For both used real-time qPCR assays, analytic sensitivity of less than 10 copy numbers per PCR reaction are reported [2 (link),24 (link)].