Research | sensitivity of PCR assays for murine gammaretroviruses | 21 May 2012

May 22, 2012


From PLoSOne, 21 May 2012.

Sensitivity of PCR Assays for Murine Gammaretroviruses and Mouse Contamination in Human Blood Samples

Li Ling Lee(1), Lin Lin(1), David S. Bell(2), Susan Levine(3), Maureen R. Hanson(1)*
(1) Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America,
(2) Department of Pediatrics, State University of New York, Buffalo, New York, United States of America,
(3) Private Practice, New York, New York, United States of America

Abstract

Gammaretroviruses related to murine leukemia virus (MLV) have variously been reported to be present or absent in blood from chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) patients and healthy controls.

Using subjects from New York State, we have investigated by PCR methods whether MLV-related sequences can be identified in nucleic acids isolated from whole blood or from peripheral blood mononuclear cells (PBMCs) or following PBMC culture. We have also passaged the prostate cancer cell line LNCaP following incubation with plasma from patients and controls and assayed nucleic acids for viral sequences.

We have used 15 sets of primers that can effectively amplify conserved regions of murine endogenous and exogenous retrovirus sequences.

We demonstrate that our PCR assays for MLV-related gag sequences and for mouse DNA contamination are extremely sensitive. While we have identified MLV-like gag sequences following PCR on human DNA preparations, we are unable to conclude that these sequences originated in the blood samples.

To download the full paper, please click HERE.

1 thought on “Research | sensitivity of PCR assays for murine gammaretroviruses | 21 May 2012”

  1. This paper is the fourth positive study to find MRVs (gamma retroviruses of murine origin) are associated with ME. The others are Grossberg, Lombardi and Lo.

    Unlike all the negative papers Hanson should be congratulated for using a clinical positive for optimizing most of her assays and not a synthetic virus (VP-62), which is not the viruses detected by Mikovits/Ruscetti, Lo/Alter and Grossberg. Every negative study has been flawed for failing to follow this basic step in assay design and cannot state with any certainty that their assay can detect a ME retrovirus when present.

    “The optimization was performed by using a sample that had resulted in amplification with the gagO/gagI nested PCR.”

    This next quote is also very sobering, for it highlights how difficult it is to detect MRVs and how much research will need to be done to identified all those infected.

    “The PCR primers that we and others have employed for screening for XMRV and MLV-like sequences will allow detection of only a subset of viruses related to MLV.”

    It is also important to realise that single round PCR never detected anything in Lombardi et al. either. A single round assay is just not sensitive enough for gamma retroviruses which exist in such a low level in the body.

    It would be very informative if the authors would state what the rate of infection was in the Bell compared to the Levine patients. Bell is know to use the Canadian criteria for his patients, but Levine uses Fukuda. As Levine is providing patients for the NIH study, not Bell, we should expect to see similar rates of infection to controls. Then the study can be repeated with the same cohort as was used in Lombardi et al. Using the Canadian criteria to select in only those with ME.

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