Detection of murine leukemia virus or mouse DNA in commercial RT-PCR reagents and human DNAs

Abstract

The xenotropic murine leukemia virus (MLV)-related viruses (XMRV) have been reported in persons with prostate cancer, chronic fatigue syndrome, and less frequently in blood donors. Polytropic MLVs have also been described in persons with CFS and blood donors. However, many studies have failed to confirm these findings, raising the possibility of contamination as a source of the positive results. One PCR reagent, Platinum Taq polymerase (pol) has been reported to contain mouse DNA that produces false-positive MLV PCR results. We report here the finding of a large number of PCR reagents that have low levels of MLV sequences. We found that recombinant reverse-transcriptase (RT) enzymes from six companies derived from either MLV or avian myeloblastosis virus contained MLV pol DNA sequences but not gag or mouse DNA sequences. Sequence and phylogenetic analysis showed high relatedness to Moloney MLV, suggesting residual contamination with an RT-containing plasmid. In addition, we identified contamination with mouse DNA and a variety of MLV sequences in commercially available human DNAs from leukocytes, brain tissues, and cell lines. These results identify new sources of MLV contamination and highlight the importance of careful pre-screening of commercial specimens and diagnostic reagents to avoid false-positive MLV PCR results.

Figure 1. Contamination of commercial AMV reverse transcriptases (RT) with MLV sequences.

A. Representative real-time, generic MLV protease (pro) amplification plot. Blue lines, XMRV RNA standard extracted from 22Rv1 cell culture supernatants from 10⁶–10⁰ copies per reaction; burgundy lines with triangles, 4/16 (25%) water only controls tested positive for MLV pro sequences using the ABI TaqMan Fast 1-step Master Mix; bright green line, RFU, relative fluorescent units. B. Representative gel image showing nested PCR detection of 208-bp MLV polymerase (pol) sequences in water only control reactions using Finnzymes RobustI AMV RT. Lanes 1–16, water only controls; lanes 17–20, XMRV RNA extracted from 22Rv1 cell culture supernatants from 10³, 10², and 10 copies per reaction, respectively; M. molecular weight marker.

Figure 2. Inference of contamination origin in commercial RT-PCR reagents and human genomic DNAs.

Phylogenetic analysis of 168-bp polymerase (pol) sequences. Bootstrap values ≥60 are shown. New sequences from the current study are shown in red (RT contaminants) and blue (human genomic DNA contaminants). Sequence names in purple are those reported as contaminants from other human studies. GenBank accession numbers for prototypical murine leukemia virus (MLV) reference sequences are provided in parentheses. XMRV clades are collapsed for presentation only and include identical sequences from CFS-WPI-1106(GQ497344), CFS-WPI-1178(GQ497343), preXMRV2(FR871850), PC-VP35(DQ241301), PC-VP42(DQ241302), PC-VP62(DQ399707), 22Rv1/CWR-R1(FN692043) The 168-bp pol sequences are available from the authors upon request. GenBank does not accept sequences less than 200-bp in length. Sequences coded as XMRV VP and WPI are from prostate cancer (PC) and CFS patients, respectively. BD; blood donor sequences. Viral host receptor tropism is indicated by blue (xenotropic), purple (polytropic), and yellow (ecotropic) spheres. PMLV, polytropic MLV; XMLV, xenotropic MLV.

Figure 3. Contamination of commercial human genomic DNA specimens with mouse DNA.

A. Representative real-time, generic mouse mitochondrial DNA and B. intracisternal A particle (IAP) polymerase (pol) amplification plots. Blue lines, mouse mtDNA or IAP plasmid DNA from 10⁶–10⁰ and 10⁷–10¹ copies per reaction, respectively; burgundy lines with triangles, human genomic DNA extracted from brain tissue of persons with various inflammatory diseases and two aliquots of the same lot of human peripheral blood lymphocytes (PBL); bright green line, RFU, relative fluorescent units. IAP and mtDNA assay are sensitive to at least 10 copies per reaction.

Figure 4. Inference of contamination origin in commercial human genomic DNAs.

Phylogenetic analysis of 301-bp gag sequences. Bootstrap values ≥60 are shown. New sequences from the current study are shown in blue (human genomic DNA contaminants). Sequence names in purple are those reported as contaminants from other human studies. GenBank accession numbers for prototypical murine leukemia virus (MLV) reference sequences are provided in parentheses. XMRV clades are collapsed for presentation only and include CFS-WPI-1106(GQ497344), CFS-WPI-1178(GQ497343), CFS-WPI-1130(GQ483508), CFS-WPI-1138(GQ483509), CFS-WPI-1169(GQ483510), CFS-WPI-CI-1303(JF907633), CFS-WPI-CI-1313(JF907643), CFS-WPI-CI-1314T(JF907644), CFS-WPI-CI-1307(JF907638), CFS-WPI-CI-1310(JF907641), CFS-WPI-CI-1327(JF907636), preXMRV2(FR871850), PC-VP35(DQ241301),and PC-VP62(DQ399707). Accession numbers for the new gag sequences generated in our study are JN629081-JN629087. Sequences coded as XMRV VP and WPI are from prostate cancer (PC) and CFS patients, respectively. BD; blood donor sequences. Viral host receptor tropism is indicated by blue (xenotropic), purple (polytropic), and yellow (ecotropic) spheres. PMLV, polytropic MLV; XMLV, xenotropic MLV.