6 ms·

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

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.

stonogo 2y ago
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.
- realityfactchex 2y ago
  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.
  - stonogo 2y ago
    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.
- timewizard 2y ago
  > is present or it is not. 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? > the standard is 30±10 cycles The difference between 2^30 and 2^40 is massive.
  - stonogo 2y ago
    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.
blackguardx 2y ago
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.
- tanseydavid 2y ago
  Kary Mullis, inventor of PCR does not agree with your analogy.
  - churchturing 2y ago
    He also * believed in astrology * denied Avogadro’s number (???) * practiced telepathy * didn’t believe in HIV causing AIDs * didn’t believe in climate change * didn’t believe in the ozone hole While he certainly had incredible intelligence, clearly him believing something isn’t a hugely convincing reasoning. disclaimer: i am a person who believes in Avogadro’s number and not astrology. truly an intellectual outcast and rebel
    - dekhn 2y ago
      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_and_the_Avogadro_constant 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.
      - churchturing 2y ago
        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
  - 2y ago

stonogo 2y ago

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.

realityfactchex 2y ago
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.
- stonogo 2y ago
  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.
timewizard 2y ago
> is present or it is not. 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? > the standard is 30±10 cycles The difference between 2^30 and 2^40 is massive.
- stonogo 2y ago
  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.

blackguardx 2y ago

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.

tanseydavid 2y ago
Kary Mullis, inventor of PCR does not agree with your analogy.
- churchturing 2y ago
  He also * believed in astrology * denied Avogadro’s number (???) * practiced telepathy * didn’t believe in HIV causing AIDs * didn’t believe in climate change * didn’t believe in the ozone hole While he certainly had incredible intelligence, clearly him believing something isn’t a hugely convincing reasoning. disclaimer: i am a person who believes in Avogadro’s number and not astrology. truly an intellectual outcast and rebel
  - dekhn 2y ago
    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_and_the_Avogadro_constant 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.
    - churchturing 2y ago
      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
- 2y ago