False negative results from using common PCR reagents

[Guest guest]

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 , 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.