PCR Master Mixes Harbour Murine DNA Sequences. Caveat Emptor!

Abstract

BackgroundXMRV is the most recently described retrovirus to be found in Man, firstly in patients with prostate cancer (PC) and secondly in 67% of patients with chronic fatigue syndrome (CFS) and 3.7% of controls. Both disease associations remain contentious. Indeed, a recent publication has concluded that “XMRV is unlikely to be a human pathogen”. Subsequently related but different polytropic MLV (pMLV) sequences were also reported from the blood of 86.5% of patients with CFS. and 6.8% of controls. Consequently we decided to investigate blood donors for evidence of XMRV/pMLV.Methodology/Principal FindingsTesting of cDNA prepared from the whole blood of 80 random blood donors, generated gag PCR signals from two samples (7C and 9C). These had previously tested negative for XMRV by two other PCR based techniques. To test whether the PCR mix was the source of these sequences 88 replicates of water were amplified using Invitrogen Platinum Taq (IPT) and Applied Biosystems Taq Gold LD (ABTG). Four gag sequences (2D, 3F, 7H, 12C) were generated with the IPT, a further sequence (12D) by ABTG re-amplification of an IPT first round product. Sequence comparisons revealed remarkable similarities between these sequences, endogeous MLVs and the pMLV sequences reported in patients with CFS.Conclusions/SignificanceMethodologies for the detection of viruses highly homologous to endogenous murine viruses require special caution as the very reagents used in the detection process can be a source of contamination and at a level where it is not immediately apparent. It is suggested that such contamination is likely to explain the apparent presence of pMLV in CFS.

Lack of evidence for human infection with Xenotropic murine leukemia virus-related virus in the Brazilian Amazon basin

“…This DNA is detectable in some batches and can thus generate false positive results. The results of the present study did not support this conclusion; the reagents used were provided by several different manufacturers, including the manufacturer used by Tuke et al 28 , and no false positives were observed. It thus seems more likely that contamination of reagents, human blood or tissue samples with murine genomic DNA was restricted to only a few research laboratories, which would account for the divergence of results.…”

“…The most likely explanation for false positive results is contamination of commercial PCR reagents [12][13][14] . Additionally, Tuke et al 28 proposed that a specifi c Taq DNA polymerase may contain monoclonal mouse antibodies with varying quantities of murine DNA. This DNA is detectable in some batches and can thus generate false positive results.…”

Phylogenetic Analysis of Murine Leukemia Virus Sequences from Longitudinally Sampled Chronic Fatigue Syndrome Patients Suggests PCR Contamination Rather than Viral Evolution

“…There is no credible hypothesis that could explain these observations in the absence of PCR contamination. It appears to be extremely difficult to do mouse-free PCR, and we note that other studies in which contamination has been demonstrated have also amplified a diverse range of MLVs (27,38). We propose that the detection of murine virus in human samples be more rigorously controlled using IAP PCR (26) to rule out murine DNA contamination and robust phylogenetic analysis to rule out random amplification of endogenous proviruses (12), which can exist at a high copy number in the genomes of mice or in cell lines that become infected with mouse viruses during routine experimentation.…”

No Detection of Xenotropic Murine Leukemia Virus-Related Viruses in Prostate Cancer in Sanandaj, West of Iran

“…Proposed reasons to explain the conflicting data are unknown but may be: technical differences, lack of standardized XMRV PCR assays, assay sensitivity, contamination by and cross-reactivity of XMRV PCR assays with closely related endogenous MLVs such as trace quantities of mouse genomic DNA found in reagents and samples (Hue et al, 2010;Oakes et al, 2010;Robinson et al, 2010;Sato et al, 2010;Knox, Carrigan et al, 2011;Tuke et al, 2011), differences in the geographical distribution of XMRV, sequence differences among XMRV genomes (Silverman et al, 2010;Singh et al, 2010;Knox et al, 2011) and factors related to the population genetic factors (Switzer et al, 2011).…”