Quantification of Torque Teno Virus and Epstein-Barr Virus Is of Limited Value for Predicting the Net State of Immunosuppression After Lung Transplantation

Nordén, Rickard; Magnusson, Jesper; Lundin, Anna; Tang, Ka-Wei; Nilsson, Staffan; Lindh, Magnus; Andersson, Lars-Magnus; Riise, Gerdt C; Westin, Johan

Nucleic acid isolation was performed with a MagNA Pure LC total nucleic acid or DNA isolation kit for serum (TTV) or whole blood (EBV and CMV) using a standardized protocol, according to the instructions (Roche Diagnostics, Mannheim, Germany). Input/output volume was set to 200/100 µL, and the sample was eluted in extraction buffer. The TTV-DNA levels were determined using a 7300 real-time PCR system (Applied Biosystems, Foster City, CA). Each PCR reaction contained 2 µL of extracted total nucleic acid, 10 µL of 2X Universal Master Mix (Applied Biosystems, Foster City, CA), 0.5 µL and 20 µM of forward and reverse primer, respectively, 0.3 µL and 20 µM of BHQ hydrolysis probe, and 2 µL of RNase-free H2O. The reaction conditions were 50°C for 2 minutes and 95°C for 10 minutes prior to 45 cycles at 95°C for 15 seconds and 60°C for 60 seconds. The assay range was determined by serial dilution of plasmids with an insert of a synthesized sequence matching the TTV PCR product, and quantification was obtained from a plot of Ct values. CMV-and EBV-DNA levels were determined using a method described previously with minor modifications [27] . All primer and probe sequences are listed in Supplementary Table 1 . For EBV and CMV quantification, a control sample consisting of unrelated Phocine herpesvirus 1 (PhHV-1) was included prior to nucleic acid extraction.