Date: January 5, 2012
Author: Ben Butkus
URL: http://www.genomeweb.com/pcrsample-prep/cdc-team-finds-widespread-murine-virus-mouse-dna-contamination-commercial-pcr-re
CDC team finds widespread Murine Virus, Mouse DNA contamination
in commercial PCR reagents
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In recent months, several independent research groups have found that
the continued discovery in human genetic samples of sequences from
murine leukemia viruses and related infectious agents is most likely a
result of contaminated commercial PCR reagents, and not, as originally
thought, a case of the viruses infecting humans.
Now, a group from the US Centers for Disease Control and Prevention
has published evidence showing that commercial RT-PCR reagents and
even human DNA preparations from several life science vendors contain
MLV or mouse DNA, suggesting that the extent of this contamination may
be much larger than anyone had previously thought.
As such, the scientists believe that their findings underscore the
need for laboratories to carefully pre-screen commercial molecular
diagnostic reagents and nucleic acid specimens to ensure they are not
contaminated with foreign DNA and to avoid potential false-positive
PCR results when testing human clinical specimens.
"If people are testing humans for these kinds of viruses, or any
viruses really, they need to pre-screen their reagents first and make
sure that they don't have some kind of external source of
contamination," William Switzer, a CDC scientists and lead author of
the recent study, told PCR Insider this week.
Since 2006, when scientists first discovered a new gammaretrovirus
called xenotropic MLV-related virus, or XMRV, and reported the
presence of XMRV sequences in samples from patients with chronic
fatigue syndrome or prostate cancer, the scientific community has
systematically debunked the link by showing that the appearance of the
MLV-like sequences was likely due to commercial reverse transcriptases
contaminated with the mouse virus DNA (PCR Insider, 12/22/11).
A few of these contrary reports even demonstrated contamination in
specific life science research tools such as Platinum Taq from Life
Technology's Invitrogen business and nucleic acid extraction columns
from Qiagen.
In the meantime, Switzer and colleagues in the laboratory branch of
the CDC's HIV/AIDS prevention division - who were part of the Blood
XMRV Scientific Research Working Group organized in December 2009 to
scrutinize the purported link between XMRV and human disease -
frequently detected low levels of MLV and XMRV-like protease sequences
in control samples or negative blood donor plasma using a qRT-PCR
assay employing the AgPath One Step RT-PCR kit, also sold by Life
Technologies.
Suspecting contamination of the RT-PCR kit with trace amounts of
residual MLV-like plasmid sequences, the group decided to further test
different AgPath kits from different lots, as well as water-only
control samples, for the presence of MLV/XMRV sequences. As detailed
in their research paper, published last month in PLoS One, they found
low levels of the mouse virus sequences in seven of 16 replicates of a
new, previously unopened AgPath kit, but found no sequences in the
water-only samples.
To further investigate the breadth of this contamination, Switzer and
colleagues then tested several other commercial RT enzymes and kits
from multiple manufacturers using MLV and XMRV RT-PCR tests developed
in their CDC laboratory.
As with the AgPath kit, the researchers discovered low levels of
either protease or polymerase sequences from MLV/XMRV in five other
commercial kits: the TaqMan 1-step master mix and TaqMan RNA-to-Ct
kits from Life Tech's Applied Biosystems business; the Brilliant II
QRT-PCR master mix from Agilent; the Transcriptor 1-step RT-PCR kit
from Roche; and the Robust I RT-PCR kit from Thermo-Fisher's Finnzymes
business.
Even more surprising, according to Switzer, was the fact the reverse
transcriptases found in some of the contaminated kits were known to be
derived from a completely unrelated virus, avian myeloblastosis virus.
"We found contamination in... enzymes that were prepared from MLV
expression vectors, which was not that surprising," Switzer said. "But
what was surprising was when we decided to check out avian
myeloblastosis virus-derived recombinant RTs, and found the same level
of contamination of MLV sequences in some of those. Maybe these
companies produce both AMV- and MLV-derived RTs, and do it in large
bioreactors... or there are sources of contamination in their
facilities, where they grow it in extremely large quantities. It's
very hard to get rid of this contamination."
Further, the CDC researchers discovered that several different
commercially available human DNAs, sold by Sigma-Aldrich and Biochain
and used for negative controls in their PCR assays, were contaminated
with mouse DNA - "some with very high levels of mouse DNA," said
Switzer.
"We contacted [Biochain] and told them about it, and they
independently confirmed those results, and went back to their
production lines and changed some things to try and reduce and
eliminate the contamination," Switzer said. "We found that they did a
good job of that, but there was still trace amounts of contamination
in the new DNA preparations that they sent us."
Since publishing their PLoS One paper, the CDC researchers have also
discovered commercial reverse transcriptases expressing plasmids that
were generated from HIV sequences, Switzer said, raising the specter
that this type of unexpected contamination may even go beyond MLV/XMRV
and has the potential to cause false positives in HIV studies - an
idea that the group is currently examining more closely.
"It's been an eye opener," Switzer said. "When we first read some of
these papers [linking XMRV with CFS or prostate cancer] we were
thinking either that this is real, or it's contamination on a scale
that's never been reported in science before. And the latter scenario
seems to be what has happened."
Switzer further hypothesized that "mice are used in a variety of
experiments around the world in many laboratories. People work with
mouse and human cell lines in the same facilities and laboratories.
Apparently there has been so much research done on MLVs, and people
have used them to develop other tools like RT-expressing plasmids,
that... it's just everywhere."
If this is the case - and evidence continues to mount suggesting that
it is - the researchers believe that all laboratories using qRT-PCR to
detect the presence of sequences from MLV/XMRV or possibly any virus
in human samples may need to rigorously screen all diagnostic reagents
and specimens for potential contamination.
"If you get these kinds of unexpected findings like people originally
reported... you really have to go back and do all the necessary
quality control experiments to make sure you're absolutely right
before you stick your neck out there and publish these kinds of
results that get refuted, and then end up retracting those papers,"
Switzer said.
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