2.6. Viral Load by Quantitative PCR (qPCR) Analysis

Viral loads were measured in tissue homogenates of skin punch biopsy samples. Total DNA was extracted from each sample using a QIAcube robot and viral genome copies were quantified via qPCR. Briefly, samples were extracted using TRI Reagent (Sigma Aldrich, St. Louis, MO, USA, Cat no# T9424) following the manufacturer’s recommendations. Viral DNA isolated from samples was eluted with nuclease-free H₂O and stored at −70 °C or below. The quantitative PCR (qPCR) reaction used to assess vaccine viral load was targeted to the Orthopox E9L DNA Polymerase gene using commercially available Pan-orthopox Virus E9L Gene-specific Quantitative PCR Assay Detection Kit (BEI Resources, Manassas, VA, USA, Cat no# NR-9350).