Author Topic: Bloody working group - Why the negative control were positive  (Read 725 times)

ISO

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Bloody working group - Why the negative control were positive
« on: September 23, 2011, 08:52:20 AM »
Little run down for you all.

In this study the WPI found 2 plasma gag samples positive.  Both were from the negative controls.

These 2 were sequenced and are 1 to 3 nucleotides different to VP62, which makes them the same type Lo et al found.


"However, two plasma clinical aliquots were reported as positive in the WPI nested RT-PCR gag assay. These samples were from two different negative controls, and only one out of the three replicates was positive in each case. Sequencing of the excised bands revealed 1-3 base changes compared to XMRV derived from 22Rv1 (supporting online text)."


Ruscetti didn't take part in this section of the study and Lo change assay, to one using VP62 primers.

If others used VP62 to validate plasmas as negative, they would not have picked up anything because plasma RNA has sequence variability.  Which explains why Lo cannot now detect the type of virus found in Lo et al.   So the VP62 assays could not find integrated MLVs. (not the mouse sort) What they can find is free floating virions in plasma.

As the WPI use lower annealing temperatures their primers would pick up both.

So I would ask, were the plasmas validated negative using VP62 as positive controls to optimise the PCR and did those assays use high annealing temperatures?  because using a free floating clone, which does not exist in nature, will never give a PCR the clinical sensitivity to pick up a methylated provirus.

High stringency primers will also not detect virion RNA in plasma because, as shown by the macaque studies, the RNA has sequence variability.

asleep

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Re: Bloody working group - Why the negative control were positive
« Reply #1 on: September 23, 2011, 02:52:05 PM »
Very important points.

I would say that the concept of a "pedigreed negative" is a farce to begin with. Conceptually and practically, it is almost impossible to conclusively show that something is truly negative. There are countless ways -- some known, some not -- in which you can fail to find something that is actually present (wrong assays, assay failure, wrong conditions, sample preparation, etc, etc). Labeling something as "pedigreed negative" after looking at it using one method (shown repeatedly to be inadequate) is nothing short of fraud.

At most, these samples can be said to be "pedigreed negative" for only the exact methodology used to pedigree them. Even saying that assumes many things (that the assays worked when the samples were pedigreed; that there is no transience of positivity across samples from the same source).

The concept of a "pedigreed positive" is tricky too, but for different reasons. The issue here -- especially with new discoveries -- is showing conclusively that you are finding what you think you are finding. However, conceptually, this is much different than "pedigreed negatives" because you only need to conclusively demonstrate one positive thing as opposed to ruling out countless negative variables. Demonstrating consistent positivity of a sample using multiple methods is usually sufficient to ensure that something is indeed positive. Even then, issues of transience and detecting near the limits could mean that a "pedigreed positive" isn't always positive.

So while both types of pedigree have conceptual hurdles, "pedigreed negatives" are significantly more difficult -- if even possible -- to ensure.

Of course the denialobots have been all over the place harping about how "there is no such thing as a known clinical positive" (i.e. "pedigreed positive"), yet they are palpably absent in the face of the far more egregious conceptual flaw of the "pedigreed negative."

Tango

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Re: Bloody working group - Why the negative control were positive
« Reply #2 on: September 23, 2011, 02:57:57 PM »
They didn't include pedigreed negatives in this part of the study.  That tells us a lot.
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