Limits...
PCR master mixes harbour murine DNA sequences. Caveat emptor!

Tuke PW, Tettmar KI, Tamuri A, Stoye JP, Tedder RS - PLoS ONE (2011)

Bottom Line: Indeed, a recent publication has concluded that "XMRV is unlikely to be a human pathogen".Methodologies for the detection of viruses highly homologous to endogenous murine viruses require special caution as the very reagents used in the detection process can be a source of contamination and at a level where it is not immediately apparent.It is suggested that such contamination is likely to explain the apparent presence of pMLV in CFS.

View Article: PubMed Central - PubMed

Affiliation: Transfusion Microbiology Research and Development, National Transfusion Microbiology Laboratories, National Health Service Blood and Transplant, Colindale, London, United Kingdom. philip.tuke@hpa.org.uk

ABSTRACT

Background: XMRV is the most recently described retrovirus to be found in Man, firstly in patients with prostate cancer (PC) and secondly in 67% of patients with chronic fatigue syndrome (CFS) and 3.7% of controls. Both disease associations remain contentious. Indeed, a recent publication has concluded that "XMRV is unlikely to be a human pathogen". Subsequently related but different polytropic MLV (pMLV) sequences were also reported from the blood of 86.5% of patients with CFS. and 6.8% of controls. Consequently we decided to investigate blood donors for evidence of XMRV/pMLV.

Methodology/principal findings: Testing of cDNA prepared from the whole blood of 80 random blood donors, generated gag PCR signals from two samples (7C and 9C). These had previously tested negative for XMRV by two other PCR based techniques. To test whether the PCR mix was the source of these sequences 88 replicates of water were amplified using Invitrogen Platinum Taq (IPT) and Applied Biosystems Taq Gold LD (ABTG). Four gag sequences (2D, 3F, 7H, 12C) were generated with the IPT, a further sequence (12D) by ABTG re-amplification of an IPT first round product. Sequence comparisons revealed remarkable similarities between these sequences, endogeous MLVs and the pMLV sequences reported in patients with CFS.

Conclusions/significance: Methodologies for the detection of viruses highly homologous to endogenous murine viruses require special caution as the very reagents used in the detection process can be a source of contamination and at a level where it is not immediately apparent. It is suggested that such contamination is likely to explain the apparent presence of pMLV in CFS.

Show MeSH

Related in: MedlinePlus

Maximum likelihood tree of gag sequences.The MLV RAxML tree for gag sequences lying between XMRV primers gagIF and gagIR was developed from the endogenous MLV sequences identified by Jern et al [19], sequences derived from CFS patients by Lo et al [5] (shown in red and blue) and the sequences amplified in this study (in green). In addition to sequences from the RNA and DNA, “1F_131010” and “1F” are included. 1F_131010 was derived from the amplification of cDNA synthesized from RNA extracted from the tissue culture fluid supernatant of an XMRV culture, kindly provided by Professor M McClure. 1F was derived from the amplification of Balb/c mouse DNA (Sigma). Bootstrap values >50 are indicated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3102076&req=5

pone-0019953-g002: Maximum likelihood tree of gag sequences.The MLV RAxML tree for gag sequences lying between XMRV primers gagIF and gagIR was developed from the endogenous MLV sequences identified by Jern et al [19], sequences derived from CFS patients by Lo et al [5] (shown in red and blue) and the sequences amplified in this study (in green). In addition to sequences from the RNA and DNA, “1F_131010” and “1F” are included. 1F_131010 was derived from the amplification of cDNA synthesized from RNA extracted from the tissue culture fluid supernatant of an XMRV culture, kindly provided by Professor M McClure. 1F was derived from the amplification of Balb/c mouse DNA (Sigma). Bootstrap values >50 are indicated.

Mentions: The results of the sequence alignments are shown as a phylogenetic tree (Figure 2) and confirm those obtained in FASTA searches. Sequences 12D, 2D and 7H fall within the pMLVs whereas 7C and 12C are mpMLVs. 3F and 9C cluster with xenotropic MLVs and XMRV. Interestingly 9C carried the same deletion as MLV001 (HQ601957), which was amplified from a patient with CFS.


PCR master mixes harbour murine DNA sequences. Caveat emptor!

Tuke PW, Tettmar KI, Tamuri A, Stoye JP, Tedder RS - PLoS ONE (2011)

Maximum likelihood tree of gag sequences.The MLV RAxML tree for gag sequences lying between XMRV primers gagIF and gagIR was developed from the endogenous MLV sequences identified by Jern et al [19], sequences derived from CFS patients by Lo et al [5] (shown in red and blue) and the sequences amplified in this study (in green). In addition to sequences from the RNA and DNA, “1F_131010” and “1F” are included. 1F_131010 was derived from the amplification of cDNA synthesized from RNA extracted from the tissue culture fluid supernatant of an XMRV culture, kindly provided by Professor M McClure. 1F was derived from the amplification of Balb/c mouse DNA (Sigma). Bootstrap values >50 are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102076&req=5

pone-0019953-g002: Maximum likelihood tree of gag sequences.The MLV RAxML tree for gag sequences lying between XMRV primers gagIF and gagIR was developed from the endogenous MLV sequences identified by Jern et al [19], sequences derived from CFS patients by Lo et al [5] (shown in red and blue) and the sequences amplified in this study (in green). In addition to sequences from the RNA and DNA, “1F_131010” and “1F” are included. 1F_131010 was derived from the amplification of cDNA synthesized from RNA extracted from the tissue culture fluid supernatant of an XMRV culture, kindly provided by Professor M McClure. 1F was derived from the amplification of Balb/c mouse DNA (Sigma). Bootstrap values >50 are indicated.
Mentions: The results of the sequence alignments are shown as a phylogenetic tree (Figure 2) and confirm those obtained in FASTA searches. Sequences 12D, 2D and 7H fall within the pMLVs whereas 7C and 12C are mpMLVs. 3F and 9C cluster with xenotropic MLVs and XMRV. Interestingly 9C carried the same deletion as MLV001 (HQ601957), which was amplified from a patient with CFS.

Bottom Line: Indeed, a recent publication has concluded that "XMRV is unlikely to be a human pathogen".Methodologies for the detection of viruses highly homologous to endogenous murine viruses require special caution as the very reagents used in the detection process can be a source of contamination and at a level where it is not immediately apparent.It is suggested that such contamination is likely to explain the apparent presence of pMLV in CFS.

View Article: PubMed Central - PubMed

Affiliation: Transfusion Microbiology Research and Development, National Transfusion Microbiology Laboratories, National Health Service Blood and Transplant, Colindale, London, United Kingdom. philip.tuke@hpa.org.uk

ABSTRACT

Background: XMRV is the most recently described retrovirus to be found in Man, firstly in patients with prostate cancer (PC) and secondly in 67% of patients with chronic fatigue syndrome (CFS) and 3.7% of controls. Both disease associations remain contentious. Indeed, a recent publication has concluded that "XMRV is unlikely to be a human pathogen". Subsequently related but different polytropic MLV (pMLV) sequences were also reported from the blood of 86.5% of patients with CFS. and 6.8% of controls. Consequently we decided to investigate blood donors for evidence of XMRV/pMLV.

Methodology/principal findings: Testing of cDNA prepared from the whole blood of 80 random blood donors, generated gag PCR signals from two samples (7C and 9C). These had previously tested negative for XMRV by two other PCR based techniques. To test whether the PCR mix was the source of these sequences 88 replicates of water were amplified using Invitrogen Platinum Taq (IPT) and Applied Biosystems Taq Gold LD (ABTG). Four gag sequences (2D, 3F, 7H, 12C) were generated with the IPT, a further sequence (12D) by ABTG re-amplification of an IPT first round product. Sequence comparisons revealed remarkable similarities between these sequences, endogeous MLVs and the pMLV sequences reported in patients with CFS.

Conclusions/significance: Methodologies for the detection of viruses highly homologous to endogenous murine viruses require special caution as the very reagents used in the detection process can be a source of contamination and at a level where it is not immediately apparent. It is suggested that such contamination is likely to explain the apparent presence of pMLV in CFS.

Show MeSH
Related in: MedlinePlus