Detection of XMRV genes from viral Ab-positive PC patients. (A) Primer positions used in the PCR assay (upper panel). Gly-Gag; homologous region to glycosylated Gag of MLVs at the NH2 terminus of Gag. RNAs purified from the plasma of viral Ab-positive PC patients (P24 and P28) were used in a nested RT-PCR with primers GAG-O-F/R and GAG-I-F/R. Unnecessary lanes between the negative control without template RNA (N) and P24 have been removed from the original image (lower panel). (B) The detection limit of nested genomic PCR. Genomic DNA extracted from serially diluted 293T cells infected with 22Rv1 cell-derived XMRV (indicated as 105~0) was mixed with genomic DNA extracted from 105 293T cells. For one reaction of PCR with a volume of 20 μl, 100 ng of each DNA mixture was used. The final concentration of viral genome contained in a PCR reaction was calculated as 7610.5-0.152 cell equivalents of genomic DNA from 293T cells infected with 22Rv1 cell-derived XMRV (corresponding to the lanes indicated as 105-10-1 of infected 293T cells). The detection limit of the nested PCR was calculated as approximately 1.5 cell equivalents (indicated as "101"). (C) Inconsistent results of nested genomic PCR tests for XMRV using genomic DNA extracted from PBMCs. In a 20 μL volume, 100 ng genomic DNA were used for amplification. Nested genomic PCRs were performed on September 17 (left) and September 18 (right), 2008. M, molecular size marker; N, negative control without nucleic acids; P24 and P28, nucleic acids purified from PBMCs of P24 or P28; HV, genomic DNA of healthy volunteer; PC, diluted XMRV VP62 plasmid; arrow head, amplified band using inner primer pair.