See also: Dr Singh's study shows that her new assays are unable to detect HGRVs in known positives
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Clifford H. Shin1, Lucinda Bateman2, Robert Schlaberg1, Ashley M. Bunker3, Christopher J. Leonard1, Ronald W. Hughen4, Alan R. Light4, Kathleen C. Light4, and Ila R. Singh1,*
1 Department of Pathology, University of Utah, Salt lake City, Utah, 84112
2 Fatigue Consultation Clinic, Salt Lake City, Utah, 84102
3 ARUP Laboratories, Salt Lake City, Utah, 84108
4 Department of Anesthesiology, University of Utah, Salt Lake City, Utah, 84112
* Corresponding author: Mailing address: Emma Eccles Jones Medical Research Building, Department of Pathology, 15 North Medical Drive East, Suite #2100, Salt Lake City, UT 84112, Phone: (801) 213-3737, Fax: (801) 585-7376, Email: ila.singh@path.utah.edu
ABSTRACT
Chronic fatigue syndrome (CFS) is a multi-system disorder characterized by prolonged and severe fatigue that is not relieved by rest. Attempts to treat CFS have been largely ineffective primarily because the etiology of the disorder is unknown. Recently CFS has been associated with xenotropic murine leukemia virus-related virus (XMRV) as well as other murine leukemia virus (MLV)-related viruses, though not all studies have found these associations. We collected blood samples from 100 CFS patients and 200 self-reported healthy volunteers from the same geographical area. We analyzed these in a blinded manner using molecular, serological and viral replication assays. We also analyzed samples from patients in the original study that reported XMRV in CFS. We did not find XMRV or related MLVs, either as viral sequences or infectious virus, nor did we find antibodies to these viruses in any of the patient samples, including those from the original study. We show that at least some of the discrepancy with previous studies is due to the presence of trace amounts of mouse DNA in the Taq polymerase enzymes used in these previous studies. Our findings do not support an association between CFS and MLV-related viruses including XMRV and off-label use of antiretrovirals for the treatment of CFS does not seem justified at present.
Page 14: In order to be confident of detecting XMRV in clinical samples, w 295 e developed our PCR assays to
296 the robust and reliable standards of clinically used assays. We developed four distinct qPCR
297 (TaqMan) assays that target different regions of the XMRV proviral sequence. One targeting the
298 pol gene region, has been used extensively by us (22) and others (15, 21, 25), and of all the
299 published PCR-based tests for XMRV, has been shown to be the most specific (8). We improved
300 on the sensitivity of this assay so that it could reliably detect at least 5 viral copies of XMRV
301 DNA (see Fig. 2A). To allow for possible variations in viral sequence in our subjects, we
302 developed three additional qPCR tests that targeted the LTR, gag and env regions of XMRV
303 proviral DNA. We characterized each of these assays in detail to determine their limits of
304 detection, specificity and reproducibility. Assay characteristics for the LTR qPCR are shown in
305 Fig. 2B. We could reliably detect less than 5 copies of XMRV plasmid DNA in a background of
306 400 ng of human placental DNA, and the assay was linear over a large range, viz. 5000 to 5
307 copies of viral DNA. This sensitivity was matched by the assays targeting the env and pol
308 regions. The gag assay was also able to reliably detect at least 5 viral copies with an average of 3
309 cycles delay in crossing the threshold (threshold cycle, Ct, see Fig. 2C). We also demonstrated
that the assays had good precision and reproducibility as demonstrated by the R2 310 values of Cts
311 being close to 1. To determine intra-run precision, 4 different amounts of XMRV plasmid DNA,
312 ranging from 5 copies to 5000 copies, were amplified in 3 different reactions in the same run. To
313 determine inter-run precision, the 4 different levels of XMRV DNA were amplified in 3 different runs on 3 different days. The assays had good intra-run precision with a mean CV of 0.99%, and also good inter-run precision, with a mean CV of 1.36%. We also verified that the tests were specific for XMRV and did not detect other common human pathogens, including other human retroviruses (Fig. 2C).
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See also: Dr Singh's study shows that her new assays are unable to detect HGRVs in known positives
1 comment:
I probably want to tell the Chronic fatigue syndrome (CFS), because my friend is puzzled with it recently. After being diagnosed, it's better to reduce the stress of surroundings.
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