2.2 HCMV DNA detection
For WB-DNAemia detection in routine diagnostic procedures, viral DNA was extracted from 200 µl of whole blood using QIAsymphony® DSP DNA Mini kit (Qiagen, Heidelberg, Germany) on the Qiasymphony platform (Qiagen, Heidelberg, Germany). Real time-PCR master mix was prepared using QuantiFast Pathogen PCR+IC KIT (Qiagen, Heidelberg, Germany) according to the manufacturer’s instructions. The sequences of the primers and probes used were previously described [9,10]. Reactions were performed on the Rotor-Gene Q thermocycler (Qiagen, Heidelberg, Germany) with the following thermal protocol: 95°C for 5 min then 45 cycles at 95°C for 15sec and 60°C for 30sec.
For PL DNAemia, 200µl of plasma was extracted using QIAamp® DSP Virus Spin kit. Real PCR assays were performed using TaqMan® Universal PCR MasterMix (Applied Biosystems, USA) and the same primers and probes set. Reactions were performed on 7300 Real time PCR system thermocycler with the following thermal protocol: 50°C for 2min, 95°C for 10min, 50 cycles at 95°C for 15sec and 60°C for 1min. To detect virion-associated PL DNAemia, plasma was treated with DNAse before DNA extraction as previously reported [3]