Screening of CFS patients using genomic PCR. (A) Primer positions used in the PCR assay. Gly-Gag; homologous region to glycosylated Gag of MLVs at the NH2 terminus of Gag. The detection limits of the genomic PCR assays with primers 419F and 1154R, and In-For 363 and Del-Rev 461 are shown in (B) and (C), respectively. Genomic DNA extracted from serially diluted 293T cells infected with 22Rv1 cell-derived XMRV was mixed with genomic DNA extracted from 1.0 × 105 293T cells. For one reaction with a volume of 20 μl, 100 ng of each DNA mixture was used. The final concentration of the detected viral genome was calculated as 7610.5-0.152 copies (corresponding to the lanes of 105-10-1 infected 293T cells, respectively) in a reaction. The detection limit of both PCR tests is approximately 1.5 cell equivalents of genomic DNA from 293T cells infected with 22Rv1 cell-derived XMRV indicated as "101"of infected 293T cells. (D) Representative results of PCR assay with primers indicated in (B) (upper) and (C) (middle). The human GAPDH gene was examined as an internal control (bottom). M, molecular size marker; HV, genomic DNA of healthy volunteers; N, no template; U, genomic DNA of uninfected 293T cells; P, genomic DNA of infected 293T cells.