Monday, November 21, 2011

Dr O'Keefe: I am not entirely convinced that MLVs aren't infecting humans

Denise S. O'Keefe, PhD. said...:

In response to Andy's comment above, well said - I agree with you with regards to XMRV and the plasmid contamination problem. However your findings have the potential to apply to related MLVs. I am not entirely convinced that polytropic or other MLVs aren't infecting humans. I think that the same level of healthy (usually) skeptism regarding positive findings of these viruses should have been extended to the negative finding papers, as well. I do not believe that it has; ill-defined levels of detection and poorly described methods attest to this.

As I mentioned in my post, we found that it was unbelievably easy to generate false-negative results using PCR detection - we accidentally found this while testing our levels of detection for mouse DNA contamination in patient samples. I think the best remedy for this is to include an internal control in every patient sample tube; the control would need to be almost identical to the intended target but ideally, a little larger (we'd take a plasmid containing the targeted region and clone in a 30 base pair piece of DNA in the middle). This would be spiked into patient samples at the level of detection of the assay. Therefore if inhibitors are present in the sample, the internal control will not amplify and the test is inconclusive. The reason for having the internal control a little larger is so it can be distinguished from a true positive when run on a gel or otherwise analyzed. If you are looking for MLV-type sequences, the sample would be spiked with a "gag" like sequence (or whichever bit is the target), and a replicate sample should be tested for mouse DNA contamination, also spiked with an internal positive control for mouse DNA (i.e. mouse mitochondrial DNA target). To my knowledge none of the published papers have used these type of controls, instead using a single tube with VP62 plasmid as a control for the entire PCR reaction. The problem with that is vanishingly small amounts of plasmid have very high copy numbers of the target, in this case XMRV/MLVs (many sets of primers against XMRV will also amplify other MLVs). Almost all the papers we looked at did not specify how much of the positive control they used; but let's just say they used one microliter of a typical plasmid prep - so 50ng of plasmid...diluted it 1:1000, so now its 0.05 nanograms of plasmid...guess how many copies you have?

For VP62 that would be about 6 million. If the test sample has significantly less copies, or differences at the primer binding sites, it might not be detected. In addition, as we demonstrated in our paper, accidental carry-over contamination - for example for touching a lab book that has contacted your gloved hand that loaded a previous PCR on a gel (we are talking less than millionths of a liter) - from a negative control or PCR reaction - is sufficient to completely block legitimate amplification of 2 million copies of an MLV GAG sequence.

Until I see those kinds of controls, I would suggest that inability to find a virus in a sample is not the equivalent of evidence against the existence of said virus.

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