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DNA extraction columns contaminated with murine sequences

Discussion in 'XMRV Research and Replication Studies' started by Firestormm, Jan 6, 2012.

  1. Firestormm

    Firestormm Guest

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    Cornwall England
    Morning, I can't see that this has been posted before. If it has please remove. The full text is available free is anyone's interested from the same link and I have pasted the 'discussion' beneath this abstract:

    Note: this was published in August 2011 and maybe it hasn't been available in full for free before but you might just want to shove it in the library or something.

    DNA extraction columns contaminated with murine sequences: http://www.ncbi.nlm.nih.gov/pubmed/21876752

    Erlwein O, Robinson MJ, Dustan S, Weber J, Kaye S, McClure MO.

    Source:Jefferiss Research Trust Laboratories, Section of Infectious Diseases, Imperial College London, London, United Kingdom.

    Abstract

    'Sequences of the novel gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV) have been described in human prostate cancer tissue, although the amounts of DNA are low. Furthermore, XMRV sequences and polytropic (p) murine leukemia viruses (MLVs) have been reported in patients with chronic fatigue syndrome (CFS).

    In assessing the prevalence of XMRV in prostate cancer tissue samples we discovered that eluates from nave DNA purification columns, when subjected to PCR with primers designed to detect genomic mouse DNA contamination, occasionally gave rise to amplification products.

    Further PCR analysis, using primers to detect XMRV, revealed sequences derived from XMRV and pMLVs from mouse and human DNA and DNA of unspecified origin. Thus, DNA purification columns can present problems when used to detect minute amounts of DNA targets by highly sensitive amplification techniques.'

    Discussion

    'There are many commercially available kits that rely on DNA binding columns to extract and purify DNA from tissues or cultured cells. Our observations by two different laboratory investigators (OE and MJR) using three different kits and working in separate laboratories, demonstrate that they can be contaminated with DNA of diverse provenance.

    This includes DNA from mice. It cannot be ruled out that some of the buffers used during the DNA extraction process were contaminated and, in turn, resulted in contamination of some of the columns. We tested the elution buffers from several kits and found no evidence for contamination. A confounding issue was the fact that tissue lysis buffers and washing buffers in these kits were found to contain substances that inhibited the PCR and, therefore, the buffers could not be reliably tested.

    Therefore, it is possible that these buffers contain traces of DNA which bind to the columns and are eluted in later steps, contaminating the sample. However, it is telling that dismantled column parts soaked in elution buffer resulted in an IAP signal while the elution buffer control did not, suggesting that the columns themselves can be contaminated.

    Recently, several publications documented that widely used PCR enzymes and buffers can be contaminated with murine DNA [13], [14], [24]. Taken together with the data presented here, these results may explain some of the spurious detections of XMRV or related pMLV sequences [2], even in laboratories that use neither mice nor XMRV-infected cell lines and avoid enzymes known to contain traces of murine DNA.

    For those involved in detecting minute amounts of retroviral sequences in human tissue, these data may serve as a useful reminder to check reagents to confirm that murine sequences are absent before analysing tissue samples.'
     
  2. alex3619

    alex3619 Senior Member

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    Hi, there are two issues arising from these contamination findings. The first is that a lot of research could be problematic, everything needs to be tested for contamination early on. The second is that when contamination is found, other means must be found to confirm or refute the presence of something like the contaminant in the test sample. The failure of a test due to contamination does not refute the existence of the target in the sample, it just means its failed to confirm it. Bye, Alex
     
  3. Firestormm

    Firestormm Guest

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    How can one rule-out contamination Alex? By proving integration sites? Phylogenetic Analysis? I wonder if a Lombardi-type paper will ever be accepted again without the reviewers demanded more evidence.

    Lessons to be learned? We could start a new thread!!
     
  4. Tony Mach

    Tony Mach Show me the evidence.

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    Upper Palatinate, Bavaria
    1. Compare what the negative XMRV studies did with regards to contamination with what the positive studies did. And then tell me with a straight face that you think the positive studies were done somehow better and should be trusted. Or that you can draw ANY conclusions from the positive studies. Or that you should not trust the negative studies. It is always moving the goal posts. First the negative studies are bad because they weren't properly done, then when it turns out that the positive studies weren't done properly it suddenly doesn't matter, because the virus could be there anyway. If you don't like the scientific method you are free to use a pendulum.

    2. Tell me that all of the 100+ scientists in over 30 studies didn't want to find a novel pathogen, or were too stupid. All of them. And then consider the alternative explanation, that if there is a virus, that it isn't a (gamma-)retrovirus.

    3. Well, there are X/P/MLV contamination and there is XMRV plasmid "contamination" to be considered. XMRV plasmid does not accidentally contaminate patient samples and it MOST CERTAINLY is NOT found in patients. There are two conclusions you can draw from this, and I leave it up to you to find a relaxing brain-fog free minute (or more) to think about this yourself.

    4. There are billions of viruses out there. Literally. And you can not rule out the existence of any in your, mine or anybodies body. But that is no proof that the virus of your choice is there (or even causes disease). You could equally pick up the Enterovirus familiy or the Herpesvirus family (both would make more sense in my view). Can you rule out that Coxsackie B causes some or most ME/CFS cases? Yeah, I didn't think so.

    (And as a side note, wouldn't it be fun if Lipkin finds Enteroviruses in people with ME/CFS. Because then you should take a hard look at the work Vincent Racaniello has been doing for decades and the vicious attacks made by some people with ME/CFS at Racaniello and virology: that they are either idiots or have an agenda and that they should look at XMRV and stop everything else.)
     
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