A Dusty Miller, Fred Hutchinson Cancer Research Center
29 December 2010
After solving the contamination problem reported in this article, do you find any XMRV RNA in sera from chronic fatigue syndrome patients in Japan?
Competing interests
None.
Sato et al missed a trick
Isabel Greenwood, PA institute
12 January 2011
A number of retrovirologists have expressed a belief that the recent discovery of MLV related viruses in people with Myalgic Encephalomyelitis also called Chronic Fatigue Syndrome (ME/CFS) and Prostate Cancer Patients are in fact contaminants. Four studies were posted simultaneously in this journal in Dec 2010 where the authors offered their interpretation of their findings as evidence in support of their hypothesis. Others have argued that alternative explanations of their findings are readily available. They argue that even without other lines of evidence, not considered in these studies, an alternative interpretation argues against the advocates of contamination argument. This letter focuses on a critique of one of these papers namely the one authored by Sato and others (1). The abstract is included for the readers perusal.
"During pilot studies to investigate the presence of viral RNA of xenotropic murine leukemia virus (MLV)-related virus (XMRV) infection in sera from chronic fatigue syndrome (CFS) patients in Japan, a positive band was frequently detected at the expected product size in negative control samples when detecting a partial gag region of XMRV using a one-step RT-PCR kit. We suspected that the kit itself might have been contaminated with small traces of endogenous MLV genome or XMRV and attempted to evaluate the quality of the kit in two independent laboratories. We purchased four one-step RT-PCR kits from Invitrogen, TaKaRa, Promega and QIAGEN in Japan. To amplify the partial gag gene of XMRV or other MLV-related viruses, primer sets (419F and 1154R, and GAG-I-F and GAG-I-R) which have been widely used in XMRV studies were employed. The nucleotide sequences of the amplicons were determined and compared with deposited sequences of a polytropic endogenous MLV (PmERV), XMRV and endogenous MLV-related viruses derived from CFS patients. We found that the enzyme mixtures of the one-step RT-PCR kit from Invitrogen were contaminated with RNA derived from PmERV. The nucleotide sequence of a partial gag region of the contaminant amplified by RT-PCR was nearly identical (99.4 % identity) to a PmERV on chromosome 7 and highly similar (96.9 to 97.6 %) to recently identified MLV-like viruses derived from CFS patients. We also determined the nucleotide sequence of a partial env region of the contaminant and found that it was almost identical (99.6 %) to the PmERV. In the investigation of XMRV infection in patients of CFS and prostate cancer, researchers should prudently evaluate the test kits for the presence of endogenous MLV as well as XMRV genomes prior to PCR and RT-PCR tests."
This analysis will now focus on two extracts from the abstract which form the foundation of the entire study.
“We purchased four one-step RT-PCR kits from Invitrogen, TaKaRa, Promega and QIAGEN in Japan. To amplify the partial gag gene of XMRV or other MLV-related viruses, primer sets (419F and 1154R, and GAG-I-F and GAG-I-R) which have been widely used in XMRV studies were employed.”
Sato et al point to the fact that these primer sets have been widely used in XMRV studies. They do not state however that these primer sets used in a one step RT-PCR or a single round PCR have never been able to detect even population levels of XMRV (3, 4) or any other MLV related viruses. Indeed Danielson et al (5) demonstrated that primer sets which could readily detect XMRV sequences in vitro could not do so in a patient sample known to be infected, by a PCR assay targeting the env gene, or serological methods. Thus these primers in studies could not have detected this RNA sequence because they could not detect a MULV sequence of any kind.
“We also determined the nucleotide sequence of a partial env region of the contaminant and found that it was almost identical (99.6 %) to the PmERV.”
Sato and others relate that they partially sequenced the env region. One must ask why they did not sequence the entire env region which would then have enabled the virus to be identified. This is most unfortunate. In fact no region of the virus which would have enabled unequivical identification of the virus was sequenced at all.
The PCR assays used by Sato could not have detected a contaminant in any XMRV study because when they were used no MLV or MLV related virus of any kind was detected. The authors here have not taken any of the available opportunities to identify the virus. It is therefore difficult so see why this study has been presented as offering evidence in support of those who argue contamination when it clearly does no such thing.
1) Eiji Sato, Rika A Furuta, Takayuki Miyazawa; An endogenous murine leukemia viral genome contaminant in a commercial RT-PCR Kit is amplified using standard primers for XMRV; Retrovirology 2010, 7:110 (20 Dec. 2010)
2) HC Groom, VC Boucherit, K Makinson, E Randal, S Baptista, S Hagean JW Gow, FM Mattes, JR Kerr, JP Stoye and KN Boshop; Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome; Retrovirology 2010, 7:10doi:10.1186/1742-4690-7-10
3) FJM van Kuppeveld, AS de Jong, KH Lanke, GW Verhaegh, WJG Melchers, CMA Swanink, G Bleijenberg, MG Netea, JMD Galama, & JWM van der Meer (2010); Prevalence of xenotropic murine leukaemia virus-related virus in patients with chronic fatigue syndrome in the Netherlands: retrospective analysis of samples from an established cohort; British Medical Journal : 10.1136/bmj.c1018
4) BP Danielson, GE Ayala and JT Kimata; Detection of Xenotropic Murine Leukemia Virus-Related Virus in Normal and Tumor Tissue of Patients from the Southern United States with Prostate Cancer Is Dependent on Specific Polymerase Chain Reaction Conditions; J Infect Dis. (2010) 202 (10): 1470-1477. doi: 10.1086/656146
Competing interests
No competing interests
lombardi et al advised not to use one step kits
Gerwyn Morris, PA institute
21 January 2011
If the Sato and others had read the original study by Lombardi et al(2009) they would have noted that the authors of that study advised(in the text) against the use of one step kits because mouse contamination had been detected in such kits. It seems strange that Sato and others have attemped to "reinvent the wheel" in their study by presenting information that was already in the public domain
XMRV in CFS subjects?
29 December 2010
After solving the contamination problem reported in this article, do you find any XMRV RNA in sera from chronic fatigue syndrome patients in Japan?
Competing interests
None.
Sato et al missed a trick
12 January 2011
A number of retrovirologists have expressed a belief that the recent discovery of MLV related viruses in people with Myalgic Encephalomyelitis also called Chronic Fatigue Syndrome (ME/CFS) and Prostate Cancer Patients are in fact contaminants. Four studies were posted simultaneously in this journal in Dec 2010 where the authors offered their interpretation of their findings as evidence in support of their hypothesis. Others have argued that alternative explanations of their findings are readily available. They argue that even without other lines of evidence, not considered in these studies, an alternative interpretation argues against the advocates of contamination argument. This letter focuses on a critique of one of these papers namely the one authored by Sato and others (1). The abstract is included for the readers perusal.
"During pilot studies to investigate the presence of viral RNA of xenotropic murine leukemia virus (MLV)-related virus (XMRV) infection in sera from chronic fatigue syndrome (CFS) patients in Japan, a positive band was frequently detected at the expected product size in negative control samples when detecting a partial gag region of XMRV using a one-step RT-PCR kit. We suspected that the kit itself might have been contaminated with small traces of endogenous MLV genome or XMRV and attempted to evaluate the quality of the kit in two independent laboratories. We purchased four one-step RT-PCR kits from Invitrogen, TaKaRa, Promega and QIAGEN in Japan. To amplify the partial gag gene of XMRV or other MLV-related viruses, primer sets (419F and 1154R, and GAG-I-F and GAG-I-R) which have been widely used in XMRV studies were employed. The nucleotide sequences of the amplicons were determined and compared with deposited sequences of a polytropic endogenous MLV (PmERV), XMRV and endogenous MLV-related viruses derived from CFS patients. We found that the enzyme mixtures of the one-step RT-PCR kit from Invitrogen were contaminated with RNA derived from PmERV. The nucleotide sequence of a partial gag region of the contaminant amplified by RT-PCR was nearly identical (99.4 % identity) to a PmERV on chromosome 7 and highly similar (96.9 to 97.6 %) to recently identified MLV-like viruses derived from CFS patients. We also determined the nucleotide sequence of a partial env region of the contaminant and found that it was almost identical (99.6 %) to the PmERV. In the investigation of XMRV infection in patients of CFS and prostate cancer, researchers should prudently evaluate the test kits for the presence of endogenous MLV as well as XMRV genomes prior to PCR and RT-PCR tests."
This analysis will now focus on two extracts from the abstract which form the foundation of the entire study.
“We purchased four one-step RT-PCR kits from Invitrogen, TaKaRa, Promega and QIAGEN in Japan. To amplify the partial gag gene of XMRV or other MLV-related viruses, primer sets (419F and 1154R, and GAG-I-F and GAG-I-R) which have been widely used in XMRV studies were employed.”
Sato et al point to the fact that these primer sets have been widely used in XMRV studies. They do not state however that these primer sets used in a one step RT-PCR or a single round PCR have never been able to detect even population levels of XMRV (3, 4) or any other MLV related viruses. Indeed Danielson et al (5) demonstrated that primer sets which could readily detect XMRV sequences in vitro could not do so in a patient sample known to be infected, by a PCR assay targeting the env gene, or serological methods. Thus these primers in studies could not have detected this RNA sequence because they could not detect a MULV sequence of any kind.
“We also determined the nucleotide sequence of a partial env region of the contaminant and found that it was almost identical (99.6 %) to the PmERV.”
Sato and others relate that they partially sequenced the env region. One must ask why they did not sequence the entire env region which would then have enabled the virus to be identified. This is most unfortunate. In fact no region of the virus which would have enabled unequivical identification of the virus was sequenced at all.
The PCR assays used by Sato could not have detected a contaminant in any XMRV study because when they were used no MLV or MLV related virus of any kind was detected. The authors here have not taken any of the available opportunities to identify the virus. It is therefore difficult so see why this study has been presented as offering evidence in support of those who argue contamination when it clearly does no such thing.
1) Eiji Sato, Rika A Furuta, Takayuki Miyazawa; An endogenous murine leukemia viral genome contaminant in a commercial RT-PCR Kit is amplified using standard primers for XMRV; Retrovirology 2010, 7:110 (20 Dec. 2010)
2) HC Groom, VC Boucherit, K Makinson, E Randal, S Baptista, S Hagean JW Gow, FM Mattes, JR Kerr, JP Stoye and KN Boshop; Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome; Retrovirology 2010, 7:10doi:10.1186/1742-4690-7-10
3) FJM van Kuppeveld, AS de Jong, KH Lanke, GW Verhaegh, WJG Melchers, CMA Swanink, G Bleijenberg, MG Netea, JMD Galama, & JWM van der Meer (2010); Prevalence of xenotropic murine leukaemia virus-related virus in patients with chronic fatigue syndrome in the Netherlands: retrospective analysis of samples from an established cohort; British Medical Journal : 10.1136/bmj.c1018
4) BP Danielson, GE Ayala and JT Kimata; Detection of Xenotropic Murine Leukemia Virus-Related Virus in Normal and Tumor Tissue of Patients from the Southern United States with Prostate Cancer Is Dependent on Specific Polymerase Chain Reaction Conditions; J Infect Dis. (2010) 202 (10): 1470-1477. doi: 10.1086/656146
Competing interests
No competing interests
lombardi et al advised not to use one step kits
21 January 2011
If the Sato and others had read the original study by Lombardi et al(2009) they would have noted that the authors of that study advised(in the text) against the use of one step kits because mouse contamination had been detected in such kits. It seems strange that Sato and others have attemped to "reinvent the wheel" in their study by presenting information that was already in the public domain
Competing interests
None declared