Screening for XMRV in patient PBMCs by PCR.

<p>PCR products were analyzed on agarose gels containing ethidium bromide. (A) Representative gels for non-nested <i>env</i> (top panel) and non-nested <i>gag</i> (bottom panel) PCRs are shown containing a set of three replicates for each of 5 HIV-1⁺ patient samples. A yellow arrow indicates the sole PCR band, from patient 103219, found to be comprised of XMRV DNA by sequencing. (B) A representative gel for nested <i>env</i> PCR is shown for the same 5 HIV-1⁺ patient samples depicted in (A). Vertical black arrows in (A) and (B) indicate lanes from patient 103219 containing either (A, bottom panel) a band comprised of XMRV sequence or (B) a band of the expected mobility for the target sequence. (m) 100 base pair molecular weight marker, (1∶10⁴) DNA from one infected cell diluted in DNA from 10⁴ uninfected cells used as template for positive control.</p

Detecting murine DNA by IAP PCR.

<p>PCR products were analyzed on 1.5% agarose gels containing ethidium bromide. (A) Sensitivity of the IAP PCR assay was determined by performing PCRs on titrations of EL4 murine cell line DNA in a background of 200 ng LNCaP DNA. One murine cell equivalent (1 eq) indicates 6 pg of EL4 DNA. XMRV-infected LNCaP (iLNCaP) and uninfected LNCaP (uLNCaP) were included as controls. (B) Screening results for 17 HIV-1⁺ patient samples. Arrow points to sample 103219, which tested positive for XMRV by non-nested gag PCR. (m) 100 base pair molecular weight marker, (EL4) 6 pg of murine EL4 cell line DNA without a background of human DNA.</p

Summary of XMRV screening results.

a<p>Fractions are: number of subjects scoring positive/total number of subjects screened.</p>b<p>Ab, antibody.</p

Primers used for screening PBMC DNA specimens for XMRV.

a<p>Location of 5′ end of forward primer target site to 3′ end of reverse primer target site on XMRV VP62 reference genome (accession no. DQ399707.1).</p

Sensitivity analysis of XMRV PCR assays.

<p>PCR products were analyzed on agarose gels containing ethidium bromide. (A) Non-nested PCR assays targeting the XMRV <i>env</i> gene (top panel) and the <i>gag</i> gene (bottom panel), and (B) a nested PCR assay targeting the XMRV <i>env</i> gene were evaluated for their ability to detect either (A) provirus in XMRV-infected PNT1A cell DNA or (B) provirus in XMRV-infected LNCaP cell DNA diluted in uninfected cell DNA. Dilutions of infected cells in uninfected cells are indicated by ratios, i.e. 1∶10⁴ indicates one infected cell diluted in 10⁴ uninfected cells. (m) 100 base pair molecular weight marker, (H₂O) water used in place of DNA template as a negative control, (u) uninfected PNT1A DNA used as template for negative control.</p