Contamination of commercial PCR reagents with mouse viral DNA

Do you use hot-start PCR in your courses? Qiagen columns?  or RT-PCR? or teach quality control? If so, you need to read this.

ResearchBlogging.org

In December 2011, Science retracted a 2009 article on an association between chronic fatigue syndrome (CFS) and the presence of a virus (XMRV).  XMRV stands for Xenotropic Murine Leukemia Virus.  Normally this virus infects mice but it's been found in human samples as well.

The paper was retracted because of difficulties in replicating the results.  One of the scientists even spent time in jail because of the contentious nature of the topic and battles over trying to reproduce the data (1).  

 

Why would some researchers be able to detect a virus and others not?  

One answer could be fraud, but there could be other explanations. 

In December 2011, researchers from the Centers for Disease Control (CDC) published a paper in PLoS One that describes one of those other explanations (2).  

 

Postive results from negative controls

The CDC researchers were trying to find out if samples from people with diseases like chronic fatigue syndrome or prostate cancer or plasma samples from blood donors contained DNA from mouse viruses like XMRV or MLV.

As with any good experiment, they included negative control samples that didn't contain any DNA.

Suprisingly, they were able to detect XMRV and MLV sequences in their negative controls.  DNA contamination has been reported before in the Invitrogen One-step RT-PCR kit and Taq enzymes.  Researchers had found that the Platinum Taq in hot start PCR had been contaminated with mouse DNA, because it was present in the mouse monoclonal antibody used to keep Taq inactive during hot-start PCR.

The CDC researchers, though, were using kits from a different company, the ABI/Ambion AgPath One Step RT-PCR kit. Those kits don't contain the same reagents that were contaminated in hot start PCR.  

They decided to test all the reagents to identify the source of the error and determined that the contamination came from the reverse transcriptase in the ABI/Ambion AgPath One Step RT-PCR kit (Cat# 4387391, Austin, Tx).

 

What other reagents are contaminated?

Identifying the contamination problem with the ABI/Ambion kit, made the CDC researchers decide to look at other kits, too.

They found murine leukemia viral (MLV) DNA contamination in RT-PCR reagents from ABI/Ambion, Promega, Roche, Agilent, and Finnzymes and contamination by mouse DNA in human DNA samples from BioChain and Sigma.

 

How did the contamination happen?

In the case of the ABI / Ambion kits, DNA sequencing was used to get more information about the PCR products from the negative controls. BLAST searches comparing the sequences of the PCR products with to GenBank found the contaminating DNA was 99% identical to MoMLV.   Since the regions of viral DNA corresponded to the regions patented vectors, they concluded that the contaminating DNA probably came from an expression plasmid vector and not the entire virus.

They also performed a phylogenetic analysis with Neighbor Joining to show that the sequences were most closely related to a virus that doesn't infect humans.

 

I'm not going to go into the other experiments that they carried out, looking at the amount of enzyme, and contamination of human DNA samples with XMRV and MLV, but they were interesting well and should spark some good discussions with your students.  

 

And in conclusion:

In the discussion, the researchers note:

We also demonstrate that commercially prepared human DNAs from a variety of clinical specimens and cell lines can be contaminated with mouse DNA likely originating from mouse tissue processing in the same facility. Unlike the situation observed for the RT kits, we showed that the level of contamination of these specimens can be high and, thus, easy to detect by assays for mouse or MLV DNA.

And conclude that:

These data reinforce the need for rigorous screening of all diagnostic reagents and specimens by methods capable of detecting trace amounts of MLV and mouse DNA, and to use phylogenetic analysis to assess the biological significance of newly detected sequences.

 

These findings and conclusions are important for us to consider in biotech training programs.  Our students should know that it can be difficult to purify enzymes away from contaminating DNA and should know how to perform the assays to detect it's presence and identify the source of the problem.

 

References:

1.  Ivan Oransky, 2011 Chronic fatigue syndrome-XMRV paper retracted by Science, completely this time.  

I highly recommend having students read this.  It's a fascinating story.

2. Zheng H, Jia H, Shankar A, Heneine W, & Switzer WM (2011). Detection of Murine Leukemia Virus or Mouse DNA in Commercial RT-PCR Reagents and Human DNAs. PloS one, 6 (12) PMID: 22205995

3.  Brandner, D. and Withers, G. (2010)  Image of mouse cells.  The Cell: An Image Library, www.cellimagelibrary.org, CIL numbers 10106, 10107, and 10108, ASCB.  

The image came from the ASCB Cell Image library.

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