Epstein-Barr Virus Stimulates Torque Teno Virus Replication: A Possible Relationship to Multiple Sclerosis

Borkosky, Silvia S.; Whitley, Corinna; Kopp-Schneider, Annette; zur Hausen, Harald; deVilliers, Ethel-Michele

Data normalization was performed using the HMBS (hydroxymethylbilane synthase) housekeeping gene reported to share the same specificity in both human and cotton-top tamarin B95-8 cell lines [101] . Beacon designer 7.9 software was used for the design of the Taqman assay for HMBS, selecting a homologous region between the human and cotton-top tamarin genome. Validation of the primers and probes and assay optimization were performed as described previously [67] . Amplification reactions of HMBS were performed in 25 ml volumes, containing 12.5 ml Taqman Universal master mix (Applied Biosystems), forward and reverse primers 0.75 ml each (10 mM), 0.75 ml 59FAM-39TAMRA-labelled probe (10 mM) and 5.25 ml water. The AmpliTaq Gold DNA Polymerase was activated at 95uC for 10 min prior to amplification through 40 cycles (15 s at 95uC and 1 min at 60uC) during which fluorescent signals were measured.