Quantification of HCMV Genomes in Mouse Spleen

Total DNA was extracted from portions of mouse spleen using DNAzol (ThermoFisher) and primers and probe recognizing HCMV UL141 were used to quantify viral genomes by quantitative real-time PCR as previously described [28 (link)]. Dilutions of purified HCMV BAC DNA were used to create a standard curve. A 1 μg sample of total DNA was added to each reaction well of TaqMan FastAdvance PCR master mix (Applied Biosystems) and samples were analyzed in triplicate on a StepOnePlus TaqMan PCR machine (Applied Biosystems) with an initial activation at 50°C for 2 min and 95°C for 20 s, followed by 40 cycles of 1 s at 95°C and 20 s at 60°C. TaqMan results were analyzed using ABI StepOne software and graphed using Prism 9.0 software.