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False negative results from using common PCR reagents

RustyJ

Contaminated Cell Line 'RustyJ'
Messages
1,200
Location
Mackay, Aust
This study directly addresses the issue of false negatives in PCR assays.

False negative results from using common PCR reagents
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.
http://www.biomedcentral.com/content/pdf/1756-0500-4-457.pdf
 

Deatheye

Senior Member
Messages
161
I dont get much of it. But basically they found something that could explain false negativ results. And also checked in which papers used a procedere where there propably caused false negatives. Also a lot of papers missing the neede Information for this analyses. Correct?
 

Bob

Senior Member
Messages
16,455
Location
England (south coast)
I dont get much of it. But basically they found something that could explain false negativ results. And also checked in which papers used a procedere where there propably caused false negatives. Also a lot of papers missing the neede Information for this analyses. Correct?

Yep, that's my interpretation as well Deatheye.


It basically takes us back to the early arguements about low copy numbers, and about using inappropriate positive controls.

And I think it demonstrates that there are so many unknown variables involved in detecting a novel virus, and so we should be careful about making premature conclusions, however many studies have been carried out.

I can't interpret how relevant this is for all of the negative studies that have been carried out, but it's an interesting piece of research, and is another example of how XMRV research maybe pushing the limits of current scientific knowledge.
 

Deatheye

Senior Member
Messages
161
So now all those papers get withdrawn cause they didnt state a cricitcal information.? After all thats what some expect from the mikovits paper cause azt was not mentioned...
Srcnr

It also interesting that pcr was always sad to be so easy everyone can do it judy just insulting others if she says you doing pcr wrong. Reading this paper pcr doesnt seem that easy and error free...
 

Jemal

Senior Member
Messages
1,031
Thanks for posting RustyJ. It's an interesting study. Also, I agree with Bob: there are still many unknown variables and the situation is very complex.
 

citybug

Senior Member
Messages
538
Location
NY
They were spiking samples with mouse dna to develop a contamination test, and their samples were coming up negative. They found a chemical used in PCR kits uracil that is used to prevent carry-over contamination prevented their dna from showing positive. An alternate chemical is available. Then they went over the XMRV in CFS studies and could not tell in some which chemical was used and it seemed likely to be used in half.


Here is the abstract which is shorter. http://www.biomedcentral.com/1756-0500/4/457/abstract
 

citybug

Senior Member
Messages
538
Location
NY
This is from their conclusions.


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 and possibly used in the remaining 5 studies 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 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.
 

RustyJ

Contaminated Cell Line 'RustyJ'
Messages
1,200
Location
Mackay, Aust
This paper seems to throw a lot of doubt on just about every negative paper. Firstly, they couldn't find enough published info in some studies, and in those they did they reckon it's doubtful. This is explosive. No one has really put the negative papers under much scruitiny.
 

Enid

Senior Member
Messages
3,309
Location
UK
Thanks for posting Rusty - I am not a scientist but this seems to leave "detection" methods wide open again.
 

Bob

Senior Member
Messages
16,455
Location
England (south coast)
This paper seems to throw a lot of doubt on just about every negative paper. Firstly, they couldn't find enough published info in some studies, and in those they did they reckon it's doubtful. This is explosive. No one has really put the negative papers under much scruitiny.

Yes, it does seem to question the validity of the negative studies, and it throws up some interesting new data surrounding detecting novel viruses at very low copy levels. But it doesn't necessarily invalidate the negative studies, because we don't know exactly what methodology they used, and this data might not apply to all of the methodologies.