An Endogenous Murine Leukemia Viral Genome Contaminant in a Commercial RT-PCR Kit is Amplified Using Standard Primers for XMRV

Laboratory of Signal Transduction, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, Japan.
Retrovirology (Impact Factor: 4.19). 12/2010; 7(1, article 110):110. DOI: 10.1186/1742-4690-7-110
Source: PubMed


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.

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    • "Moreover mouse DNA in common laboratory reagents such as PCR buffers and RT-PCR kits gave false positive results for XMRV.88,89,90,91 Some commercial taq polymerases are prepared with use of a mouse monoclonal antibody, and trace amounts of DNA from the mouse hybridoma cells presumably resulted in positive PCR signals for XMRV.91,92 "
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    ABSTRACT: Xenotropic murine leukemia virus-related virus (XMRV) was discovered in 2006 in a search for a viral etiology of human prostate cancer (PC). Substantial interest in XMRV as a potentially new pathogenic human retrovirus was driven by reports that XMRV could be detected in a significant percentage of PC samples, and also in tissues from patients with chronic fatigue syndrome (CFS). After considerable controversy, etiologic links between XMRV and these two diseases were disproven. XMRV was determined to have arisen during passage of a human PC tumor in immunocompromised nude mice, by activation and recombination between two endogenous murine leukemia viruses from cells of the mouse. The resulting XMRV had a xentropic host range, which allowed it replicate in the human tumor cells in the xenograft. This review describes the discovery of XMRV, and the molecular and virological events leading to its formation, XMRV infection in animal models and biological effects on infected cells. Lessons from XMRV for other searches of viral etiologies of cancer are discussed, as well as cautions for researchers working on human tumors or cell lines that have been passed through nude mice, includingpotential biohazards associated with XMRV or other similar xenotropic murine leukemia viruses (MLVs).
    Emerging Microbes and Infections 04/2014; 3(4):e. DOI:10.1038/emi.2014.25 · 2.26 Impact Factor
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    • "However, gammaretroviruses are known to induce cancer in animals, understanding XMRV or related MLV infections in human prostate cancer tissues will shed light on their potential contribution to human disease. Proposed reasons to explain the conflicting data are unknown but may be: technical differences, lack of standardized XMRV PCR assays, assay sensitivity, contamination by and cross-reactivity of XMRV PCR assays with closely related endogenous MLVs such as trace quantities of mouse genomic DNA found in reagents and samples (Hue et al., 2010; Oakes et al., 2010; Robinson et al., 2010; Sato et al., 2010; Knox, Carrigan et al., 2011; Tuke et al., 2011), differences in the geographical distribution of XMRV, sequence differences among XMRV genomes (Silverman et al., 2010; Singh et al., 2010; Knox et al., 2011) and factors related to the population genetic factors (Switzer et al., 2011). Due to public health and medical consequences of potential XMRV or related MLVs infection in humans, we considered it is important to confirm or reject their association with prostate cancer in Iranian context. "
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    ABSTRACT: Background: Multiple etiologies have been hypothesized for prostate cancer, including genetic defects and infectious agents. A recently reported gamaretrovirus, xenotropic murine leukemia virus-related virus (XMRV) has been reported to be detected in prostate cancer. However, this virus has not been detected in similar groups of patients in other studies. Herein, we sought to detect XMRV in prostate cancers and benign controls in Sanandaj, west of Iran. Materials and methods: In a case-control study, genomic DNA was extracted from formalin fixed and paraffin embedded prostate tissues from a total of 163 Iranian patients. We developed a conventional and a nested PCR assay using primers targeting to an env specific sequence of XMRV. PCR assays were carried out on 63 prostate cancers and 100 benign prostate hyperplasias. Results: Beta-actin sequences were successfully detected in the DNA extracts from all prostate tissues, confirming DNA extraction integrity. We did not detect XMRV in samples either from prostate cancers or benign prostate hyperplasias using XMRV specific primers. Conclusions: We conclude that in our population XMRV does not play a role in genesis of prostate cancer.
    Asian Pacific journal of cancer prevention: APJCP 11/2013; 14(11):6929-33. DOI:10.7314/APJCP.2013.14.11.6929 · 2.51 Impact Factor
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    • "Later, XMRV DNA was also presumably detected in blood samples from patients diagnosed with chronic fatigue syndrome (CFS) [2]. It has then been shown that mouse DNA present in trace amounts in various PCR kits resulted in false positive XMRV PCR reactions [3,4]. Since then, published studies addressing XMRV prevalence applying well-controlled PCR protocols to prostate cancer [5,6], CFS [7] or diagnostic samples from other disorders like e.g. "
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    ABSTRACT: The gammaretrovirus termed xenotropic murine leukemia virus-related virus (XMRV) was described to be isolated from prostate cancer tissue biopsies and from blood of patients suffering from chronic fatigue syndrome. However, many studies failed to detect XMRV and to verify these disease associations. Data suggesting the contamination of specimens in particular by PCR-based methods and recent reports demonstrating XMRV generation via recombination of two murine leukemia virus precursors raised serious doubts about XMRV being a genuine human pathogen. To elucidate cell tropism of XMRV, we generated replication competent XMRV reporter viruses encoding a green fluorescent protein or a secretable luciferase as tools to analyze virus infection of human cell lines or primary human cells. Transfection of proviral DNAs into LNCaP prostate cancer cells resulted in readily detectably reporter gene expression and production of progeny virus. Inoculation of known XMRV susceptible target cells revealed that these virions were infectious and expressed the reporter gene, allowing for a fast and highly sensitive quantification of XMRV infection. Both reporter viruses were capable of establishing a spreading infection in LNCaP and Raji B cells and could be easily passaged. However, after inoculation of primary human blood cells such as CD4 T cells, macrophages or dendritic cells, infection rates were very low, and a spreading infection was never established. In line with these results we found that supernatants derived from these XMRV infected primary cell types did not contain infectious virus. Thus, although XMRV efficiently replicated in some human cell lines, all tested primary cells were largely refractory to XMRV infection and did not support viral spread. Our results provide further evidence that XMRV is not a human pathogen.
    PLoS ONE 09/2013; 8(9):e74427. DOI:10.1371/journal.pone.0074427 · 3.23 Impact Factor
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