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]