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>>>> 20 November 2011 <<<<
Editorship : j.van.roijen@chello.nl
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Official O'Keefe Lab blog & (other) cool stuff
Hello! you have reached the official blog spot of our
lab which is based at the University of Pittsburgh,
Department of Urology - our main area of study is
prostate cancer, nutrition, and epigenetics - but we
also study changes in gene expression in
benign-prostatic hyperplasia - we have made this
blog so as we can share thoughts about the lab,
papers that are just published and anything else
remotely relevant at any time, and from anywhere!
FRIDAY, NOVEMBER 18, 2011
so let's get this straight; CFS patients don't have
XMRV or MLVs, but if they did, it would explain the
neuromuscular pathology....
Dusty Miller, greatly respected in the retroviral
community, has just published a paper in the
Journal of Virology describing how, if it really
existed in humans, XMRV could induce apoptosis
of human neuroblastoma cells - presenting a
potential mechanism for the neuromuscular
pathology seen in patients with chronic fatigue
syndrome.
I don't normally blog about XMRV or other MLVs
that might be capable of infecting humans - but we
did just publish a paper (http://j.mp/v3JNKm) -
(~jvr: see below: Discussion and Conclusions) showing
how easy it would be to get false-negative results
when attempting to PCR amplify the GAG region of
viruses like these, which would be especially
relevant if the titer or copy number of the virus in
various tissues was low.
While several journals considered this scientifically
worthy work, they all thought the "interest" in this
paper would be low. By publishing in an open
access journal, we've now had 850 accesses to our
paper in 3 weeks - suggesting at least some
people are in fact interested in it.
I then wonder if anyone actually did find
polytropic/xenotropic MLVs in human disease,
would they be able to publish it?
Would it not get held back by virtue of the already
published negative data, which in most cases was
poorly controlled for or maybe not the appropriate
source of tissue?
Just a thought.
It just seems highly coincidental that the virus
(family) that apparently is so ubiquitous in the lab -
from reported contamination in heparin blood tubes
to DNA extraction kits... can in some cases infect
human cells... and can reproduce the symptoms
seen in CFS patients, whom at one point were
thought to carry the virus.
Its just a thought...you can read the paper, an epub,
here (http://j.mp/rLn3fs) - Xpr1 is an Atypical
G-protein Coupled Receptor that Mediates
Xenotropic and Polytropic Murine Retrovirus
Neurotoxicity
````
The full text of *False negative results from using
common PCR reagents* is attached for private
members, but can also be found at:
http://j.mp/v3JNKm
Discussion and Conclusions
As these results could potentially explain some of
the discrepancies in amplification of MLV-related
viruses from different laboratories, we surveyed
these publications to attempt to determine if the
authors used UNG-containing master-mixes.
Of concern, despite the importance of the actual
PCR methods used to amplify these potentially
novel viruses, we were unable to determine the
type of Taq or master-mix used in 11 of 38
publications.
Of the remaining 27 publications, 13 studies used
Taq or master-mixes likely containing UNG (it was
present in 8 studies [2-9] and possibly used in the
remaining 5 studies [10-14] that we examined).
Therefore contamination at either the individual
sample level or in the PCR reaction itself could
have inhibited amplification of legitimate target,
especially if the target is found at low levels.
Additionally, as shown in Figure 2, regardless of the
presence of UNG, PCR can be inhibited if there is
even a minute amount of contamination from
previous (negative) PCR reactions.
Based on our data, we could speculate that low
levels of PCR product contamination may not be
sufficient to significantly block amplification of the
positive control in these reactions, especially if the
positive control is the target cloned into a plasmid
and present at a high copy number.
In the majority of the studies commented on in this
manuscript, the positive control used was an
XMRV plasmid, and most papers were not clear
about the amount of plasmid used for control
amplification.
Therefore, amplification of the high copy number
positive control could occur while samples positive
for a low copy number virus could be inhibited by
carry-over PCR contamination.
To circumvent the potential for PCR inhibition, a
positive control included in each PCR reaction is
necessary to confirm that the PCR is not inhibited.
To accomplish this, a synthetic target template with
identical primer-binding sites but different size
could be constructed, and spiked into each test
sample.
This would result in potential amplification of two
bands: one band to detect the target DNA and the
other to detect the synthetic target.
In addition, the copy number of the synthetic target
spiked into the test samples would need to be at
the level of detection of the assay, so as any
inhibition would result in lack of amplification.
None of the 38 MLV-related publications we
examined used internal PCR controls.
Given the data presented in this manuscript, we
would propose that in cases such as the debate
regarding the existence of novel infectious viruses,
failure to find a virus is not the equivalent of
evidence against its existence.
The potential for false negative results needs to be
considered and carefully controlled in PCR
experiments, just as the potential for false positive
results already is.
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