I am so sick of this crap. BUT! A group in China made a really neat Real-Time PCR strategy for detecting XMRV, so Im still gonna write about it 🙂
Taqman Real-Time PCR is one of my most favoritest things ever. PCR is ‘specific’, in that we use primers designed to detect specific sequences in a sample. But its not really all that specific. The primers you use dont need to match up perfectly to the sequence you are interested in. Scientists capitalize on this to introduce mutations, or to fish around for all sequences that are ‘kinda like’ what they are interested in (like how many different places has KoRV inserted into a koalas genome), etc.
This is a problem when you really are looking for something specific, though. The gels in the last XMRV paper look like shit because of non-specific primer binding and amplification.
How do you *know* what you are seeing is what you are looking for? Taqman probes. You add a third primer to your PCR reaction that should recognize sequence in between your two original primers. When DNA polymerase hits the third primer, its like a train hitting a Honda parked on some railroad tracks. The third primer gets chopped up, releasing a fluorescent reporter that a computer reads. If you have non-specific primer binding, but the probe doesnt recognize sequence in between your primers, then the computer doesnt see it. All those non-specific bands in those crappy gels? They disappear.
You can add lots of primers with different colored probes in one reaction (called multiplexing) to look for several things in the exact same sample (I look for two different reporter genes, HIV-1, and beta-globin in one Real-Time PCR reaction).
And, Real-Time is much more sensitive than regular PCR. You know, you see those bands in gels? For the human eye to see even the faintest band, you have to have like a bazillion copies of sequence amplified. For Real-Time? Its a computer looking for those little fluorescent blips. It can literally see one copy amplified. In this paper, they got down to being able to detect 10 copies of XMRV in one milliliter of sample. To put this into perspective, I regularly generate HIV-1 stocks at 1,000,000 IU/ml. Their method is sensitive.
In this paper, they used primer-probe sets targeted towards a rather conserved region of gag. If you BLAST their primer-probes, you not only get XMRV as top hits, but also the ‘human MLV’ sequences entered by Alters group. While they only talk about XMRV in this paper, they also exclude the sequences Alter found.
They also worked out a neat way to make sure there wasnt anything wrong with their DNA– a competitive internal control. Normally, we use beta-globin or GAPDH or actin or some kind of normal cellular gene as a control in PCR. But those are non-competitive. Primers have different properties and different specificities– your control might be fine, while your experimental is f-ed up. So they created a plasmid identical to XMRV, with an altered probe binding site to spike into patient samples. So, the primers would still amplify the same sequence on the plasmid as in XMRV, but if the XMRV probe couldnt bind, the computer wouldnt see this internal competitive control. If they added an appropriate probe (with a different color), then they could ‘see’ the competitive internal control. If they got any XMRV+ samples, they could take away the XMRV probe and add the competitive control probe and make sure the signal disappeared. If the competitive internal control didnt work, then they know their PCR was fucked up, and they wouldnt detect XMRV even if it was there. I have never seen this used, and I think its wonderfully clever.
They also used appropriate groups– 1) CFS patients, 2) healthy blood donors, 3) sick but should have no connection to XMRV/CFS patients (HIV, HTLV, hepatitis patients). We were literally just talking about the importance of group #3 in the last post.
Theres only so much you can say about a negative paper, but I wanted to give this group props for making a pretty ingenious system for detecting and monitoring XMRV in a quantitiatve manner. As Ive said before, even assuming XMRV exists and is pathogenic, it is pointless to prescribe antiretrovirals to ‘infected’ patients until we have a way to quantify their efficacy.