XMRV infection was assessed by extracting DNA from 400 μL of whole blood with the Maxwell 16 System, according to the manufacturer’s instructions (Promega, Madison, WI, USA), using 500–750 ng of DNA as PCR template. The presence of virus was determined by using the protocol described in part by Lombardi . This includes a nested PCR and single-step TaqMan real-time PCR, both designed on XMRV gag. In the nested PCR, a 413-bp fragment was amplified in the first PCR. This was carried out for 35 cycles with sense primer GAG-O-F (5′-CGCGTTCGATTTGTTTTGTT-3′) and antisense primer GAG-O-R (5′-CCGCCTCTTCTTCATTGTTC-3′), under the following conditions: melting at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 45 s. The product of this reaction was then reamplified for 35 cycles with internal primers GAG-I-F (5′-TCTCGAGATCATGGGACAGA-3′) and GAG-1-R (5′-AGAGGGTAAGGGCAGGGTAA-3′) under the same PCR conditions except for 54°C for annealing. The reactions were carried out in a 50-μL PCR mixture containing Taq DNA polymerase, each dNTP at a concentration of 12.5 mM, primers (20 pmol/μL each), and optimized buffer components. All samples were tested at least in duplicate and on different occasions. The amplified product was analysed by electrophoresis on an agarose gel after ethidium bromide staining. Amplicon size was compared with standard molecular size markers. XMRV presence was also evaluated by a single-step TaqMan real-time PCR, designed on a 83-nucleotide fragment of gag and recently developed in our laboratory. Briefly, the assay was performed in a 25-μL format containing 0.9 μM each primer (Q445T, 5′-GGACTTTTTGGAGTGGCTTTGTT-3′; Q528R, 5′-GCGTAAAACCGAAAGCAAAAAT-3′), 0.1 μM labelled probe (F480PRO: FAM-ACAGAGACACTTCCCGCCCCCG-BHQ), 5 μL of extracted viral DNA, and a Master Mix containing dNTPs and Taq DNA polymerase. The reaction was run in triplicate for each sample in a iQCYCLER real-time PCR detection instrument (Bio-Rad Laboratories, Hercules, CA, USA), with a previously standardized program (95°C for 10 min; 55 cycles of 95°C for 15 s, and 60°C for 60 s). PCR data were collected and analysed with the iQ5 optical system software, version 2.1, developed by Bio-Rad. To exclude carryover contamination, negative controls were added during the DNA extraction and PCR amplification steps. To validate the amplification process, positive controls obtained from supernatants of a constitutively XMRV-infected B-cell line were run in each PCR. The assay sensitivities were measured by testing quadruplicates of ten-fold dilutions of the XMRV-positive control. The real-time PCR was found to have a sensitivity (about 100 DNA copies per millilitre of whole blood) of at least a dilution higher than that of nested PCR.