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PCR false negatives in the imperial college method

Discussion in 'XMRV Research and Replication Studies' started by Gerwyn, Feb 13, 2010.

  1. Gerwyn

    Gerwyn Guest

    There are real problems using PCR in whole blood samples because they containi inhibitors of the process and lead to losses of thebases in the target DNA Whole blood was used in the Imperial college study.

    A major problem with diagnostic and forensic techniques based on PCR is the false-negative reactions or low sensitivity caused by inhibitory substances that interfere with PCR (1, 2, 3). Of particular clinical importance is the PCR analysis of blood samples, which represents the largest fraction of human health related tests for diagnosis of genetic diseases, virus and microbial infections, blood typing, and safe blood banking. Various studies indicate that the inhibitory effect of blood on PCR is primarily associated with direct inactivation of the thermostable DNA polymerase and/or capturing or degradation of the target DNA and primers. It has been reported that the protease activity in blood also contributes to the reduced efficiency of PCR (1-5, 7, 10, 12).

    The blood resistance characteristics of the thermostable DNA polymerases vary with the source of the enzyme (6). Widely used thermostable polymerases like Thermus aquaticus DNA polymerase (Taq) and AmpliTaq Gold are completely inhibited in the presence of 0.004-0.2% whole human blood (vol/vol; 3, 4, 6). Various agents have been tested for reducing the inhibitory effect of blood on Taq. It was found that an addition of betaine, bovine serum albumin, the single-stranded DNA binding protein of the T4 32 gene (gp 32), or a cocktail of protease inhibitors can partially relieve the blood inhibition and allow Taq to work in up to 2% blood (vol/vol), although this effect could be sample specific (3, 8, 9, 11).

    Several major inhibitors of PCR in human blood have been characterized such as immunoglobulin G, hemoglobin, lactoferrin and excess of leukocyte DNA (4, 7, 10). The IgG, hemoglobin, and lactoferin have been purified from plasma, erythrocytes and leukocytes, respectively, using size-exclusion and anion-exchange chromatography (4, 7). The heme has been reported to inactivate the Taq polymerase by binding to its catalytic domain (10), while the mechanism of action of the other inhibitory components is more poorly understood. The inhibitory effect of IgG can be reduced when this plasma fraction is heated at 95 C. before adding it to PCR, or with the addition of excess non-target DNA to the PCR mixture. However, heating of IgG together with target DNA at 95 C. was found to block amplification. Inhibition by IgG may be due to an interaction with the single-stranded DNA fraction in the target DNA. The inhibitory effect could be removed also by treating the plasma with DNA-agarose beads prior to amplification (4).

    Other complicating factors include EDTA and heparin, used as anti-coagulants, which can also inhibit DNA amplification. The addition of heparinase has been shown to counteract the heparin-mediated inhibition (13, 14). Therefore, various laboratory procedures of sample preparation have been developed to reduce the inhibitory effect of blood. The DNA purification methods suitable for PCR can include additional steps like dialysis, treatment with DNA-agarose beads or Chelex 100 resin, multiple DNA washes, or a combination of dilution with buffer which causes lysis of red blood cells, centrifugation to recover the white blood cells, washing with NaOH and the addition of bovine serum albumin (2, 3, 15-19).

    These pre-treatment steps of the blood samples are generally time-consuming, labor-intensive, and can be sample specific. The guanidinium thiocianate method for DNA isolation is not suitable for reliable detection of Mycobacterium tuberculosis in clinical samples. An alternative method of DNA purification with protease K treatment followed by phenol-chloroform extraction has to be employed to relieve the inhibition (20). Separation with a QIAamp kit followed by dialysis with a Millipore filter are required for eliminating the heme inhibition of hepatitis B virus detection (21). In addition, some the above steps carry a risk of target DNA losses and are not suitable for automation. Moreover, even commercial kits specially formulated for DNA purification from blood samples such as QIAmp or GeneReleaser are not always satisfactory. The reason is due to an incomplete removal of Taq inhibitors, which can result in false-negative results. For example, 14% of the human blood samples tested for hepatitis B virus yielded false-negative results
  2. Knackered

    Knackered Guest

    Thanks for your posts Gerwyn, could you put this in laymen's terms for us with foggy brains and little knowledge of this kind of thing.
  3. Gerwyn

    Gerwyn Guest

    There are chemicals in whole blood that dramatically hamper the PCR process by stopping enzymes that the process depends on from working and or breaks up the DNA/RNA that you are looking for.So even if the base sequence you are looking for exists in the sample you cant find it and get a negative result.You can try "cleaning up" the blood to get rid of these inhibitors but the processes involved can also cause false negatives again by damaging the DNA you are looking for.There are newer processes which have varying successes in overcoming this problem .Crucially the imperial college study didn,t use them
  4. Knackered

    Knackered Guest

    Thanks Gerwyn. I really appreciate it.
  5. julius

    julius Watchoo lookin' at?

    Gerwyn, would this be 'common knowledge' for those using PCR?

    In other words, would the IC team likely have been aware of this fact?
  6. George

    George Guest

    Gerwyn Rocks

    Yes, IC defiantly knew about this problem from the start. Any researcher who has been in a room with a PCR machine knows it. It's what her Erv-ness tried to accuse the WPI of doing. It's also what Dusty Miller, Phd. brought up on the PLoS One comments section.

    You know I've been thinking about what you said before Gerwyn (yep that was like a week ago, but I'm kinda slow.) I think that the UK is broke. And it's kinda the US's fault cause China threw all that money into our financial system. (grins) So if the UK just denies, denies, denies until the US and say Germany and Canada and Australia do all the work and get the drug trial all up and running then hey. . . they won't ever actually have to pay any benefits. They just wait till the drugs are ready to go to market, cut a deal with the drug companies, give all the ME'rs the drugs and tell em to get back to work now!

    So in answer to your question, yeah, I think you're right, it's a conspiracy. Not so much to deny what will end up being proven anyway but to save money (millions of pounds). I mean heck ME/CFS are already so abused they probably figure whats a couple more years. (YACK!)
  7. Adam


    Sheffield UK
    George Rocks 2

    My mind has travelled in the same direction, George. When you look at 'our' shameful record on Gulf War Syndrome, it is easy to see how the authorities can use this as a cornerstone for CFS/ME. We looked into it guys - nothing doing. And then years later, oh yeah, looks like we overlooked a few things. Sorry guys.

    Gerwyn - please keep posting. You and all the other boffy types who know one end of a test tube from the other are keeping my spirits up.
  8. Gerwyn

    Gerwyn Guest

    yes any virologist would know about it because it is such a common problem

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