Mouse DNA contamination in human tissue tested for XMRV

Abstract

BackgroundWe used a PCR-based approach to study the prevalence of genetic sequences related to a gammaretrovirus, xenotropic murine leukemia virus-related virus, XMRV, in human prostate cancer. This virus has been identified in the US in prostate cancer patients and in those with chronic fatigue syndrome. However, with the exception of two patients in Germany, XMRV has not been identified in prostate cancer tissue in Europe. Most putative associations of new or old human retroviruses with diseases have turned out to be due to contamination. We have looked for XMRV sequences in DNA extracted from formalin-fixed paraffin- embedded prostate tissues. To control for contamination, PCR assays to detect either mouse mitochondrial DNA (mtDNA) or intracisternal A particle (IAP) long terminal repeat DNA were run on all samples, owing to their very high copy number in mouse cells.ResultsIn general agreement with the US prevalence, XMRV-like sequences were found in 4.8% of prostate cancers. However, these were also positive, as were 21.5% of XMRV-negative cases, for IAP sequences, and many, but not all were positive for mtDNA sequences.ConclusionsThese results show that contamination with mouse DNA is widespread and detectable by the highly sensitive IAP assay, but not always with less sensitive assays, such as murine mtDNA PCR. This study highlights the ubiquitous presence of mouse DNA in laboratory specimens and offers a means of rigorous validation for future studies of murine retroviruses in human disease.

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

“…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. We thank Jonathan Stoye for sharing unpublished observations.…”

“…These authors suggested that they could confirm human infection of MLV by PCR amplifying a variety of MLV sequences from the blood of CFS patients as well as healthy controls. A PCR test for mouse mitochondrial DNA was used to control for contamination with mouse DNA and found to be negative, but recently, more sensitive intracisternal type A particle (IAP)-based PCR tests for murine contamination reveal that in some cases mouse contamination is not detected by amplification of mitochondrial DNA (26). Surprisingly, Lo et al's study did not find XMRV but found a set of MLV sequences almost identical to known endogenous nonecotropic gammaretroviruses of mice.…”

No Evidence of Murine-Like Gammaretroviruses in CFS Patients Previously Identified as XMRV-Infected

“…The infection of humans with these viruses is controversial. Investigators evaluating independent cohorts of CFS patients have failed to detect XMRVor other MLVs (7-12), and contamination of human clinical material (13,14) and reagents (e.g., Taq polymerase) (15) with mouse DNA containing MLV-like sequences has been reported.To investigate these discrepancies in a more direct manner, we performed an extensive virological evaluation of blood samples from two human populations with a clinical diagnosis of CFS (16), many of whom had been diagnosed previously as XMRV-infected. The first (P1) consisted of 41 CFS patients ranging in age from 5 to 73 years who came from a private medical practice (Sierra Internal Medicine, Incline Village, Nevada).…”

“…The infection of humans with these viruses is controversial. Investigators evaluating independent cohorts of CFS patients have failed to detect XMRVor other MLVs (7)(8)(9)(10)(11)(12), and contamination of human clinical material (13,14) and reagents (e.g., Taq polymerase) (15) with mouse DNA containing MLV-like sequences has been reported.…”

XMRV ist nicht human-pathogen und hat keine Bedeutung für die Sicherheit von Blut und Blutprodukten

“…Meist waren diese Methoden jedoch nicht in der Lage zwischen endogenen retroviralen Sequenzen im Mausgenom und XMRV zu unterscheiden. Mehrere Arbeitsgruppen, die positive XMRV-Befunde erhalten hatten, konnten zeigen, dass ihre positiven Ergebnisse auf Kontaminationen mit muriner genomischer DNA zurückzuführen sind [16,17]. Die Quelle der Verunreinigung konnte in einigen Fällen identifiziert werden [15].…”