2.3 HCMV UL21.5 mRNA detection
To evaluate the presence of UL21.5 mRNA, nucleic acids were extracted from 600µl of plasma using ELITe InGenius® SP1000. Realtime RT-PCR was performed using CMV RNA ELITe MGB® kit (kindly provided by ELITech Group) targeting the UL21.5 gene, according to the manufacturer’s instructions. Nucleic acids extraction, UL21.5 cDNA synthesis and amplification and results interpretation were performed on the ELITe InGenius® platform (ELITech Group, USA). To obtain a reliable quantification, standard samples (concentrated 102-104 copies/reaction) were tested once every 60 days, following the manufacturer’s instructions. Results were expressed in copies/ml. Samples in which UL21.5 mRNA was detected but at levels below the quantification limit (30 copies/ml) were assigned an arbitrary value of 10 copies/ml. Negative samples were reported as “undetectable”.