XMRV letter \ article posted in PNAS today, reply from Dr. Lo \ Alter

LJS

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Chronic fatigue syndrome: Xenotropic murine leukemia virus-related virus, murine leukemia virus, both, or neither?
1. Otto Erlwein, 2. Steve Kaye, 3. Mark Robinson, and 4. Myra McClure
Lo et al. (1) report murine leukemia virus (MLV)-related sequences in peripheral blood mononuclear cell DNA and plasma RNA derived from patients with chronic fatigue syndrome (CFS). This follows a series of articles (2–5) that failed to confirm the detection of xenotropic murine leukemia virus-related virus (XMRV) sequences in CFS, as reported by Lombardi et al. in October 2009 (6).

Similar to Lombardi et al. (6), Lo et al. (1) found retroviral sequences in a high proportion (86.5%) of CFS cases compared with healthy controls (6.8%). The sequences detected were not XMRV, but several variant murine leukemia virus (MLV) sequences unrelated to XMRV. Throughout, Lo et al. (1) rigorously controlled for mouse DNA contamination. …
http://www.pnas.org/content/107/43/...id=1&usestrictdates=yes&resourcetype=HWCIT&ct



Response:
Reply to Erlwein et al. and Martin: On detection of murine leukemia virus-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors
1. Shyh-Ching Lo, 2. Natalia Pripuzova, 3. Bingjie Li, 4. Anthony L. Komaroff, 5. Guo-Chiuan Hung, 6. Richard Wang, and 7. Harvey J. Alter
We thank Erlwein et al. (1) and Martin (2) for their comments on our recent publication (3). As both letters raise some similar issues, we will address all of the comments here in one response.

We were aware that the SuperScript III One-Step RT-PCR System (cat. no. 12574–030; Invitrogen) had come under question for contamination with mouse DNA. For that reason, we rigorously examined the Platinum Taq polymerase we used (cat. no. 10966–034, lot 727463; Invitrogen) for any possible mouse DNA contamination. First, as stated in the article (3), no murine leukemia virus (MLV)-related gene amplicon was produced in more than 300 negative controls tested in parallel. Second, we tested the polymerase and our PCR assay system repeatedly using the highly sensitive mouse DNA-specific seminested PCR targeting the mitochondria DNA described; we never detected any mouse DNA contamination.

As for the “nonspecific” side bands seen in the gel, we have found that virus gag gene-specific primer sets could sometimes amplify human DNA nonspecifically and produce amplicons of different sizes. Thus, Martin was correct that any murine retroviral sequences would need to compete for the primers with the nonspecific reactions also occurring with normal human DNA. This would likely lower …
http://www.pnas.org/content/107/43/...id=1&usestrictdates=yes&resourcetype=HWCIT&ct


Anyone with full access, can you give us a in-depth summery?
 

Sasha

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Hi LJS - this has been out a little while and there is already a thread on it, here. All of it over my head, as usual!