Contamination of human DNA samples with
mouse DNA can lead to false detection of
Brendan Oakes1,2, Albert K Tai1, Oya Cingöz3,4, Madeleine H Henefield1, Susan Levine5, John M Coffin3,4,
Brigitte T Huber1*
Background: In 2006, a novel gammaretrovirus, XMRV (xenotropic murine leukemia virus-related virus), was
discovered in some prostate tumors. A more recent study indicated that this infectious retrovirus can be detected
in 67% of patients suffering from chronic fatigue syndrome (CFS), but only very few healthy controls (4%).
However, several groups have published to date that they could not identify XMRV RNA or DNA sequences in
other cohorts of CFS patients, while another group detected murine leukemia virus (MLV)-like sequences in 87% of
such patients, but only 7% of healthy controls. Since there is a high degree of similarity between XMRV and
abundant endogenous MLV proviruses, it is important to distinguish contaminating mouse sequences from true
Results: DNA from the peripheral blood of 112 CFS patients and 36 healthy controls was tested for XMRV with
two different PCR assays. A TaqMan qPCR assay specific for XMRV pol sequences was able to detect viral DNA from
2 XMRV-infected cells (~ 10-12 pg DNA) in up to 5 μg of human genomic DNA, but yielded negative results in the
test of 600 ng genomic DNA from 100,000 peripheral blood cells of all samples tested. However, positive results
were obtained with some of these samples, using a less specific nested PCR assay for a different XMRV sequence.
DNA sequencing of the PCR products revealed a wide variety of virus-related sequences, some identical to those
found in prostate cancer and CFS patients, others more closely related to known endogenous MLVs. However, all
samples that tested positive for XMRV and/or MLV DNA were also positive for the highly abundant intracisternal
A-type particle (IAP) long terminal repeat and most were positive for murine mitochondrial cytochrome oxidase
sequences. No contamination was observed in any of the negative control samples, containing those with no DNA
template, which were included in each assay.
Conclusions: Mouse cells contain upwards of 100 copies each of endogenous MLV DNA. Even much less than one
cell’s worth of DNA can yield a detectable product using highly sensitive PCR technology. It is, therefore, vital that
contamination by mouse DNA be monitored with adequately sensitive assays in all samples tested.
XMRV (xenotropic murine leukemia virus-related virus)
is a novel gammaretrovirus that was identified in 2006
in 10% of prostate cancers . Its functional significance
was implied by the recent observation that it is preva-
lent mainly in more aggressive tumors . In 2009, it
was reported that 67% of chronic fatigue syndrome
(CFS) patients had this infectious gammaretrovirus,
while only a small fraction of healthy volunteers was
XMRV-positive . These data were received with
enthusiasm because they pointed to a possible infectious
etiology of CFS, a chronic disability that is clinically ill-
defined. However, several research groups challenged
these conclusions almost immediately [4-11] because
they could not detect the predicted PCR products or
antibodies in cohorts of CFS or prostate cancer patients
(reviewed in [12-15]).
* Correspondence: firstname.lastname@example.org
1Department of Pathology, Tufts University School of Medicine, 150 Harrison
Avenue, Boston, MA 02111, USA
Full list of author information is available at the end of the article
Oakes et al. Retrovirology 2010, 7:109
© 2010 Oakes et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Recently, sequences related to other murine leukemia
viruses (MLVs) were reported in 80% of CFS patients ver-
sus only a small percentage of healthy controls . This
finding implicated different retroviruses specifically linked
to this patient population than the originally described
XMRV . The similarity of such sequences to large num-
bers of endogenous MLVs present in any mouse strain
[17-19] complicates interpretation of detection of such
sequences in clinical studies since possible contamination
of the human samples with mouse DNA [14,20] has to be
rigorously ruled out to validate such results.
Our laboratory has been involved in CFS research
since 2005 and has a substantial library of samples
stored from a cohort of patients and controls. Using a
nested PCR for XMRV, we detected one XMRV-like
and various MLV-like sequences, but also observed a
100% correlation between samples that were positive for
XMRV/MLV sequences and those positive for mouse
DNA, while most samples negative for XMRV/MLV
were also negative for mouse DNA. These results imply
frequent laboratory contamination with minute and
highly variable quantities of mouse DNA.
We analyzed a library of 111 stored DNA samples that
had been collected from the peripheral blood mononuc-
lear cells (PBMC) of CFS patients in 2005 for an unre-
lated project (see Methods section for description). In
addition, we collected 37 blood samples (one CFS and
36 healthy controls) in 2009-2010.
TaqMan qPCR specific for XMRV did not reveal positive
The original XMRV results from patients with prostate
cancer and CFS were obtained using a sensitive nested
PCR assay for XMRV [1,3] that also detects endogenous
MLV sequences in murine genomic DNA. These data
were later extended, employing a qPCR assay specific
for a region in the XMRV pol gene not cross-reactive
with any sequence known to be present in mouse DNA
[2, Singh, personal communication]. To test our cohort
for the presence of XMRV sequences, we analyzed
PBMC DNA with this 2ndqPCR assay, using the pri-
mers and probe as described in . Titration of DNA
from an XMRV-positive lymphoblastoid cell line, WPI-
1282 (kindly provided by the Whittemore Peterson
Institute (WPI)), resulted in detection of XMRV down
to 10-12 pg, equivalent to two cells, in the presence or
absence of 5 μg control DNA isolated from the human
LnCaP cell line (Figure 1). However, no positive
response (Ct> 60) was obtained with DNA from 112
CFS patients and 36 healthy controls, when tested at
600 ng to 5 μg per reaction (data not shown). These
data indicated that our samples were either XMRV-
negative or had more divergent MLV sequences than
originally described [1,3]. In the latter case, the qPCR
assay used, which is sensitive to small sequence differ-
ences, would not have allowed detection.
Nested PCR for XMRV gag yielded a high frequency of
To explore the possibility that XMRV sequences in
humans are more divergent than previously reported,
we used the nested PCR assay for XMRV gag sequences
mentioned above, which also detects many endogenous
MLV proviruses, as described . A preliminary titra-
tion experiment revealed that MLV-like sequences could
be detected in 2-3 pg of WPI-1282 DNA, equivalent to
<1 cell, when mixed with 200 ng control DNA (see
above) (Figure 2). This assay was used to test DNA in
triplicates of 200 ng each from our CFS and control
cohorts. Surprisingly, a high proportion of DNA samples
from the healthy volunteers (19/36), but only 2/112 of
the CFS patients, yielded PCR products of the correct
size, as tested on an agarose gel. None of the “no tem-
plate” control samples, included in each assay at least in
triplicate, gave positive results. These data suggested
that XMRV-related viruses may be highly prevalent in
the human population, but no special link of these
viruses to CFS patients was indicated. While all the
blood samples were processed in the Huber laboratory,
it should be noted that the CFS cohort mainly consisted
0 0.51 1.52 2.5
WPI 1282 in
Figure 1 Sensitivity of TaqMan qPCR for IN region in XMRV
pol. Titration of DNA from WPI-1282 (1.7, 16.7, and 166.7 cell
equivalents) in the absence (square, solid line, slope = -3.14) or in
the presence of 8.3 × 105cell equivalents of genomic LNCaP DNA
(circle, dotted line, slope = -3.04). 1.7 cell equivalents of WPI-1282
genomic DNA is detectable in 8.3 × 105cell equivalents of
background DNA. Samples were run in duplicates. All qPCR
reactions were run for 60 cycles. Samples that did not produce a
signal after 60 cycles were assumed negative for XMRV. Ct = Cycle
Oakes et al. Retrovirology 2010, 7:109
Page 2 of 10
List of abbreviations
CFS: Chronic Fatigue Syndrome; FCS: fetal calf serum; IAP: intracisternal A-
type particle; MLV: murine leukemia virus; MPLV: modified polytropic MLV;
PBMC: peripheral blood mononuclear cells; PBS: phosphate buffered saline;
PMV; polytropic MLV; WPI: Whittemore Peterson Institute; XMRV: xenotropic
murine leukemia virus-related virus; XMV: xenotropic MLV.
We would like to thank Drs. WM Switzer (CDC) for communicating the
unpublished information on the TaqMan qPCR for cox2 and JA Mikovits
(WPI) for providing the WPI-1282 lymphoblastoid cell line. The work was
supported by a grant from the HHV6 Foundation of America to BH and
grant R37 CA 089441 to JMC. JMC was a Research Professor of the American
Cancer Society with support from the FM Kirby Foundation.
1Department of Pathology, Tufts University School of Medicine, 150 Harrison
Avenue, Boston, MA 02111, USA.2Pharmacology Program, Tufts University
School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA.
3Department of Molecular Biology and Microbiology, Tufts University School
of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA.4Genetics
Program, Tufts University School of Medicine, 150 Harrison Avenue, Boston,
MA 02111, USA.5Private Practice, 115 East 72nd Street, New York, NY, USA.
BTH, AKT and BO conceived and designed the study. AKT, BO and MHH
carried out the experiments. SL collected samples from the CFS patient
cohort. AKT, BO, MHH, OC and JMC analyzed the data. BTH drafted the
manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Received: 1 November 2010 Accepted: 20 December 2010
Published: 20 December 2010
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Cite this article as: Oakes et al.: Contamination of human DNA samples
with mouse DNA can lead to false detection of XMRV-like sequences.
Retrovirology 2010 7:109.
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