This isn't my field but I always feel it's disingenuous to show PCR results without telling me how many cycles of PCR you did. PCR is effectively a magnifying glass and you're obscuring the level of magnification you used to get a detection. It doesn't seem, on it's own, to ever be a useful piece of information.
For a diagnostic application, the cycle count doesn't really matter. Either the DNA your primers are targeting is present or it is not. That's why they talk about a "positive" or "negative" PCR test -- the presence or absense of the viral DNA is a binary.
For diagnostic use like this, the standard is 30±10 cycles, but it doesn't really matter if it's ten or a hundred, since all you're really doing is verifying the presence of the target genome.
Is a robust citation each available for each of those related claims?
I'm not looking for dogmatic assertions, I mean the fundamental and reproducible papers proving those claims.
Because those are all in direct opposition with the parent. And two views of an issue will not be resolved by each claiming opposite dogma.
Hopefully someone can present the evidence. A textbook citation is not enough. Are there really controlled experiments showing these things? Or is it mostly theoretical? Is it time to ask what we know--are these "facts we have been taught", or have we looked into the scientific validity of the proofs with the criticality of a practicing scientist?
I think that very solid citations here would actually go a long way to inform the parent, but I doubt that anything short of that would do so.
I do not have the time or the inclination to perform a comprehensive literature review for the benefit of a web forum. The body of work in this area is massive, well understood, and public. You can replicate results in any university microbiology course. Thousands of students do these things every day. You can even construct your own PCR setup from a cd rom drive and parts and do it at home. Reagents and primers are available on the open market.
If, given all that, the difference between acceptance and rejection is some links in a forum post, there wasn't really any interest in the truth to start with.
Yes. And don't you use a /threshold/ mechanism to detect presence? Or you have tests so sensitive that a /single/ molecule or equivalent will deliver a reliably positive signal? I mean if it's that good why are you even doing PCR amplification in the first place?
Yes, if the DNA target is present, it will be replicated. If it is not, it will not. The point is to see whether that happens (positive result) or not (negative result). If, after PCR, there is a shitload of viral DNA in your assay, you know the virus was present in the sample. You compare assays between pre and post PCR samples to determine this.
The difference between a shitload and a bigger shitload reflects cycle count but not the nature of the result.
PCR is a basic tool. It amplifies, much like your car stereo. If you can hear music when tuning in an AM station, you don’t question the automatic gain correction the stereo is doing internally. PCR is the same way. If the target is present, it is detectable. If it isn’t then it isn’t. There are so many variables at play that the number of cycles isn’t very meaningful.
You run PCR for cycles. You run it for 32 cycles? You've got a 4 billion times multiplication of the input signal. You see the problem? "Detectable" isn't a single ended specification with "PCR." You really do need to disclose how many cycles you ran.
Similarly there's ionizing radiation in your home. Right now. Flowing through you. If it's not above the background it's not interesting or material. So just saying "we found radiation" is equally meaningless unless you tell me that level in relation to something.
he didn't deny avogadro's number so much as say that its definition was somewhat arbitrary.
IIRC he made the comment before the modern redefinition of SI units. See https://en.wikipedia.org/wiki/2019_revision_of_the_SI#Mass_a... for more details about the ongoing issues associated with absolute numeric counts of elements and their association with mass.
this is useful context, thanks. would edit my prior comment but it is no longer editable. after some more research it seems his comments on the matter were reasonable
Is sample contamination more impactful (likely to lead to a false positive) at 100 or 200 cycles than at 20? I remember some distrust of high PCR cycle counts during COVID but I never quite understood the fear.
If there is contamination with the target sequence then it will be found by the PCR analysis. I don’t know the practical limits of sensitivity but theoretically it can detect a single occurrence of the target sequence.
With enough cycles, dead virus you're successfully fighting off can be detected. It doesn't need contamination. For what it was used for, those were false positives that inflated the counts.
