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science of PCR vs. Culture test?

Discussion in 'XMRV Research and Replication Studies' started by fresh_eyes, Dec 3, 2009.

  1. fresh_eyes

    fresh_eyes happy to be here

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    Hi science-y types. So I think in other threads I've gotten an understanding of what PCR testing entails - thank you! And my understanding of the culture test is that they they basically take the sample and see if it can infect another cell line, right?

    So here's the question. Seems that someone in the poll is PCR- but Culture+. How is that possible?
    I thought the Culture only served to reconfirm a +PCR.
    Or are they using (in layperson terms) different drops of blood for these tests, and in the PCR they got a drop with (to use the fishing analogy) no fish in it, but the Culture drop did have a fish in it?
    Would a test like this be considered positive, or inconclusive?
    (Turned into a bunch of questions:eek:...thank you!)
  2. fresh_eyes

    fresh_eyes happy to be here

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    OK, I think Kurt just answered my questions on the "Inconclusive" thread:

    "I definitely can not clarify what WPI is doing. But I do know that this just seems backwards. The PCR is the more accurate test. so the way I would expect this to work is that you run the antibody test first, that is the least 'refined' test, in fact it is for MLV, which I assume they know is cross-reactive with XMRV.

    Of course this brings up the issue that if MLV antibodies are cross-reacting with XMRV, what else to they respond to?

    Anyway, if you have a positive on the MLV antibody, you run the PCR to confirm if it is an active infection. IF you do NOT have a positive the first time you run the MLV antibody, you run a culture study to amplify the sample, then run an antibody study again. If that is positive then you run the PCR.

    The PCR is the most accurate, or it should be."

    That's what I was getting at. Thanks, indirectly, Kurt!:)
    Does anyone else have a different understanding on this?
  3. Jimk

    Jimk

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    Yeah, he got culcha...

    Part of this depends on whether they activate the virus, do something to get it to actively replicate, in order to culture it. My understanding of the XMRV retrovirus is that in nonreplicating mode it is simply part of the DNA of infected host cells and in the Science research they chemically activated cells to induce replication. They also noted that viron particles in frozen serum could be activated into infectious mode.

    PCR is usually very specific, i.e. it tests for very specific protein sequences. If this sequence is not included in virons but only in actively replicating viruses, then it would not be found in a blood sample if the virus is in non-replicating mode.

    So you'd have to know more about how they induce culturable XMRV from a blood sample, and what sequences are being measured in PCR to know why you might find this difference.

    The key is that PCR is very sensitive but sometimes too specific, as well as takes very high level of training to produce a good test result. Agreement between labs for PCR can often be lousy. The statement that "PCR is more accurate" is a commonly held belief that is... not accurate to my understanding. It is more sensitive and specific for exactly what it is measuring, but can be less accurate if there is poor sampling, the primers used don't cover the right proteins, it is more subject to lab errors, etc.. That is precisely why multiple forms of tests are used to cross-validate each other: you may get indications in one form and not another.

    Culture for a lot of organisms is very difficult, and relies on getting the right sample and correct procedures to get the organism to replicate. Antibody tests are less specific but are often very sensitive to measuring the body's response to an infection. They get fooled by incompetant immune system where response is inadequate, and are prone to false negatives so a negative finding does not mean an infection isn't there.
  4. fresh_eyes

    fresh_eyes happy to be here

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    Great info JimK! I think I'm getting it!

    So:

    The advantage of PCR is that it is very, very specific - you actually "see" the virus and identify it by its genetic material. The disadvantage of PCR is that, if the retrovirus is not "activated" (meaning actively replicating?) in the cells in a sample, PCR would likely not detect it.

    This is addressed by the Culture test, which chemically activates the virus, in case it was present but not activated, and thus not detectable in the PCR.

    The advantage of the Antibody test is it's very sensitive - it shows that the body has encountered the virus, without having to find the virus itself. The disadvantage is it's not very specific, because the same antibody might be produced in response to a range of viruses, and also if your immune system isn't working well, you might not produce the expected antibodies.

    Do I have that right, more or less?

    Now, how could you be Culture+ and PCR-? Wouldn't they have to PCR the virus that they had Cultured, in order to identify it?
  5. Mithriel

    Mithriel Senior Member

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    HIV can only be found when it is actively replicating. They use this in my OHs lab to see how well antivirals are working for HIV patients.

    They have said that XMRV replicates very slowly so I would expect a lot of people to be negative for actual virus and positive for antibodies.

    Since you have retrovirus for life being antibody positive is important.

    Mithriel
  6. fresh_eyes

    fresh_eyes happy to be here

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    Seems that way to me too, Mithriel, and wasn't that borne out by the WPI study, with PCR at 67% and Antibody at 95%? Perhaps that's why the Culture is an important part of the testing - they have to force it to replicate before they can detect it?
  7. Koan

    Koan Be the change.

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    Many, many thanks for this. Even I am beginning to understand :eek:

    Based on how I am currently feeling, compared to how I sometimes feel, my assumption would be that, if I host the virus, it is not presently active.

    When I am in a financial position to consider testing, this information will be invaluable to me!

    With much gratitude to all the clever people!
    Koan

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