Gebruik makend van diverse PCR-technieken
Bell en kollega's
relatief vaker en genetische (gag)-variaties van een
in het bloed van ME/CVS-patiŽnten (in vergelijking met gezonde proefpersonen).
De gevonden virus-variaties verschilden in relatief grote mate van 22Rv1 XMRV.
Vervuiling van laboratoria en materiaal gebruikt in laboratoria met de 22Rv1-cellijn
wordt verondersteld de oorzaak te zijn van de vondst van "XMRV"
(klik hier en
Omdat de resultaten (positief/negatief) soms niet consistent waren in de tijd,
durven de auteurs geen harde conclusies te trekken over de herkomst van "MLV".
De auteurs kunnen, ondanks vele voorzorgsmaatregelen, mede daarom geen antwoord
geven op de vraag of "MLV" daadwerkelijk aanwezig is in het bloed van patiŽnten...
Uit onderstaand commentaar van
blijkt dat zij en haar collega's recent (twee varianten van) een nieuw (?)
ontdekt hebben in goedaardige prostaat-hyperplasie.
Enkele belangrijke citaten uit de studie:
One group of participants was recruited by David Bell, M.D., Lyndonville, New York,
where an outbreak occurred in 1984-1986.
The cohort contained
10 individuals who are severely ill with CFS,
10 individuals who fulfilled Fukuda criteria at one time
but now consider themselves recovered, and
20 individuals who have never been diagnosed with CFS (controls).
Susan Levine, M.D., provided samples from
20 CFS patients and 4 healthy controls
visiting her practice in Manhattan, New York.
12 controls who have never been diagnosed with CFS
were recruited from Ithaca, New York.
In addition to the nested gagO/gagI primers
utilized by Lombardi et al.  and
Lo et al. ,
we designed another set of primers for
gagL PCR (from the gag leader region)
for use in single round, 45-cycle PCR
in order to minimize
the possibility of environmental contamination with mouse sequences
that might occur during the manipulations needed for nested PCR.
PCR products corresponding to gag sequences were obtained
from whole blood or PBMC DNA from 6 different samples with single-round gagL PCR.
When nested gagO/gagI PCR was performed,
4 samples resulted in fragments corresponding to gag sequences.
5 samples from severe patients,
2 from recovered CFS patients, and
3 from control subjects
resulted in detection of gag PCR products.
Samples from one severely ill and one recovered patient
exhibited PCR products with both gagL and gagO/gagI amplifications.
All of these samples were negative in mouse mtDNA assays.
MLV-like gag sequences detected in LNCaP cultures
Two samples gave
gagO/gagI PCR products: 11F2-P4 and 14F2-P4.
With gagL PCR,
7 samples were positive:
2F1-P6, 4F1-P6, 9F1-P6, 10F3-P6, 11F2-P4, 12F2P6, and 13F3-P6.
5 additional samples irreproducibly gave
gagO/gagI PCR products and
11 sometimes were PCR positive for gagL;
due to the inconsistency, we decided to score these as negative.
gag PCR of DNA from PBMCs of control subjects in Ithaca, New York
The trees illustrate that
the sequences amplified from the samples of this study
were related to polytropic MLV-like sequences, and
that all gagL sequences were distinct from 22Rv1 XMRV,
which contains deletions in the gag leader region.
Nested PCR analysis of our initial batch of 30 samples
resulted in a significant difference in frequency of gag PCR products
between patients and controls;
however, continued analysis failed to maintain this association.
Sensitivity of PCR assays for murine gammaretroviruses and mouse contamination in human blood samples.
PLoS ONE 7(5): e37482. doi:10.1371/journal.pone.0037482.
Lee LL, Lin L, Bell DS, Levine S, Hanson MR.
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.