The LabCorp. Sarscov2 is using up to 40 cycles. Can anyone stop this madness?
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Rookie numbers, got to cycle up those positive!
CASEdemic must go on!!
And when they want the results for the vaccine they lower the cycles. It is complete madness.
A small but important correction: they will lower the number of cycles that counts as a positive (or increase the threshold value). Easy to do, too: in an update to the protocol, add a statement along the lines of: "due to revision to procedures, Ct value of 20 counts as positive, as opposed to 25 in earlier version." Or, hell, just put in on the instructions of the newly distributed kits.
So those are cycles to threshold. Fair enough. What is needed, as the barest minimum, to make sense of those, is how many cycles to threshold count as a positive? If it is 20-25, grudgingly, 30, I can accept those results as sort of valid. Beyond 30, fuggedabouitit. This alone says that half of those samples are false positives, or, at least, questionable.
They are using 38 for n1 and n2 and 40 for rnase
Right, and they may have a legit reason for that, such as quality control. What I question is how many Cts count as a "case".
Lemme explain here. Ct figure is simply the number of times you have to do melt-anneal-extend cycle to generate enough DNA to reach a certain pre-determined level (threshold). That figure is backwards-related to the amount of virus in your sample: the more virus you have, the fewer Cts you'll need.
Based on what we know about PCR, it works reliably when you do 20-25 cycles. It works a lot less reliably if you do 30. Beyond 30, it becomes a mess, but sometimes you have to do it to hunt a very rare target; then you go out of the way with your QC/QA, which, I bet, they don't do. I can make some assumptions and show my work, if needed. If you are past 35 cycles in a clinical assay, it's a catch-22: either you are running a perfect assay and picking up medically insignificant amounts of viral DNA, or you are running a sloppy assay and picking up false positives.
So, my question is, how many (or how few) Cts count as a case? In other words, where is that mark on the X axis, beyond which you begin to reject positives as false? If it is 20-25, then OK, but that tells you that about half of the cases reported should be disregarded as false positives. If it is beyond 30, this is where things get unreliable.
And I guarantee you, if one wanted to manipulate the number of cases, the total number of cycles would remain the same. It is the number of cycles to threshold (or the threshold value itself) that would be manipulated: if you lower the Ct value cutoff that counts as a positive (from 25 to 20, for example), you'll detect fewer "cases" in the same population. Same effect is produced by increasing threshold setting on the machine.
https://www.fda.gov/media/136151/download
In Fig. 1 they count 33.2 curve as a positive? I call bullshit: that kink around cycles 27-29 hints at nonspecific priming.
Here is your smoking gun:
They do not do positive controls. No negative controls either, or I am not seeing them. I am sorry, this is not acceptable even for a third-rate journal, much less for making life-and-death decisions.
Even this paper says that at the limits of detection they are using it is probably not virus that can self replicate and that the link between these detections and infectivity in humans is not known. I cant believe that the canadians are arguing that cycle count shouldnt be reported.
Here is your SECOND smoking gun:
It is not reproducible.
Very useful paper, thanks!