...I don't think most of them thought, however, that culture was going to play such a major role; that is another of XMRV's surprises....With the technology available now I'll bet they thought they would have a straight shot at it. Indeed nothing in the Science paper suggested that they wouldn't - the WPI used a normal PCR test to look for XMRV and they found it...
...They should be able to find it some of the time using ordinary PCR.....because that's what the WPI did - the WPI did straight nested PCR to find the gag sequence; that was their major finding - and they were using the standard prostate clone to do it I believe; it's just necessary to use culture to find it everywhere it is...There is something different with the nested PCR's that everyone is using...
I honestly can't believe we are still where we are....NOBODY expected this; the WPI, NCI and Cleveland Clinic thought XMRV would just sail through - a few confirmatory studies would validate their findings and then we'd be off doing treatment studies in six months......its amazing!!!!
There had to be reasons that nobody had stumbled across this in the past, even those who weren't concerned with CFS. Accidentally finding a new retrovirus in humans should have been a major discovery long ago. (Yes, I'm well aware of DeFreitas' work. She thought she was chasing a delta retrovirus. Had she been supported well enough to resolve the confusion she might have found this. Here I'm talking about others.)
One clarification here, "straight" or "direct" PCR is a conversational term for something different from "nested" PCR, and this distinction is central to the dispute. The first stage of nested PCR amplifies a lot of junk. The second stage carefully picks out specific sequences. Varying the primers and conditions has enormous effect on sensitivity and selectivity. Used carelessly nested PCR can produce bizarre results.
One discovery during the last year, (which has yet to influence these trials,) was that, at least in some cells, enzymes called APOBEC3 'hypermutate' inserted provirus. This is a natural defense against retroviruses; other mechanisms may remain undiscovered. Essential human genes are either not mutated by this, or are repaired by other cell mechanisms. The virus takes advantage of redundancy in the genetic code to use codons which will be converted to synonyms by these mutations. The result is that the vast majority of sequences inserted by the virus are not letter-for-letter copies of the reference viral sequence, which PCR typically requires.
Because mutated sequences with substitutions leading to synonyms will produce the same sequence of amino acids in polypeptides, and the same proteins, these different sequences will produce the same virus. APOBEC3 mutates inserted DNA sequences, not the RNA sequences the virus uses for its genes. Some resulting virions will likely be competent to reproduce, and may be identical to the unmutated virus.
This makes a stage of culturing virus essential to detection. Culturing has been specifically left out of these trials. How WPI was ever able to detect virus without it puzzled me. My tentative answer is that they chose patients with active infections, where immune cells infected by the virus were naturally activated and already reproducing to combat it. This fits the peculiar finding that the strongest evidence of virus often comes from people who are asymptomatic. If their immune system shows a healthy response to infection, something equivalent to culturing is going on. Newly inserted provirus has yet to be hypermutated. (This may also be relevant to the discussion about delayed processing.)
Comments about any small number of false positives have to consider the wildly improbable things that happen when people are rushed and the situation is confusing. (We have plenty of reason to believe there has been confusion.) Some cases come down to such absurdities as applying a label for the wrong sample, or mixing up the box with guaranteed-clean sample containers with those still untested. If the sample is contaminated near the source it may say nothing about the lab. If you use large numbers of samples, the probability some will be false positives goes up. If you stick with small numbers, the effect of a single error with be exaggerated.
(I can testify to similar mix ups in a completely different context where expenses are very high and development speed is important. In trying to trace the cause of failures in new electronics for important projects, one report I saw revealed the integrated circuit inside a package was an entirely different device from the one labeled on the outside. Avoiding these blunders is one aspect of the problem which raised the cost of generic ICs from 40 cents to $100 in some high-reliability applications. This seems absurdly excessive, unless you learn that those components control nuclear weapons. "All in favor of economy measures raise your hands." Eliminating these was neither cheap nor easy.)
Funding for crash programs researching questions pertinent to national survival runs into the billions, with much wasted in retrospect. This question has not been funded that way. Many avoidable mistakes will be obvious in later analysis.
Research on anything new is constantly chasing a moving target. Not only are many labs having to play catch-up out of the starting gate, they are competing with greyhounds who have nearly caught the hare. Expecting quick resolution in these circumstances is a recipe for disappointment.
. That's a good point. Well, that should prod them to work even harder to figure out what's going with the straight PCR......if they accept the culture tests then it must be there - so find it! 
