Artus cmv pcr kit

Snap-frozen tissue samples were processed with a blood DNA isolation kit (Qiagen, Hilden, Germany). DNA concentrations were determined by spectrophotometry. For detection of HCMV DNA, 20 μL of the DNA samples was used for PCR analyses with the Artus^® CMV TM PCR kit according to the manufacturer's instructions (Qiagen). Quantitative real-time PCR (RT-qPCR) was performed and analyzed with a StepOnePlus-PCR cycler (Thermo Fisher). C_T values were used to calculate the number of copies of HCMV genomes by a standard dilution curve provided with the kit and adjusted to the respective DNA concentrations. For detection of BBL-gB-CAR, 7.5 μL of the DNA samples were used for PCR analyses with TaqMan Universal Mastermix II (Applied Biosciences, Vilnius, Lithuania), 900 nM each forward and reverse primers, and 250 nM DNA probe with 5′-FAM/3′-TAM label (Eurofins, Ebersberg, Germany; sequences: forward: 5′-AGCTGCCGATTTCCAGAAGA-3′; reverse: 5′-GCGCTCCTGCTGAACTTCA-3′; Probe: [FAM] 5′-AAGGAGGATGTGAACTGAGA-3′ [TAM]). RT-qPCR was performed as described above. A standard dilution curve was measured using the retroviral plasmid DNA.

Tissues were processed with the Blood and Tissue isolation kit (Qiagen, Hilden, Germany). DNA concentration was determined by spectrophotometry and stored at −20°C. Twenty microliter of the DNA sample was used for PCR analyses with an Artus® CMV TM PCR kit according to the manufacturer's instructions (Qiagen). The standard curve was determined with primers specific for HCMV sequences and internal control primers against a human gene. PCR was performed and analyzed with StepOnePlus-PCR cycler (ThermoFisher). The Ct values were used to calculate the copies of HCMV genomes per sample adjusted with the respective DNA concentrations. For detection of HCMV in human lymphocyte subsets, cells were sorted using a FACS Aria 2 (BD Bioscience).