An endogenous murine leukemia viral genome contaminant in a commercial RT-PCR kit is amplified using standard primers for XMRV

Abstract

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.

Figure 1

Amplification of MLV-like viral sequences in Kit I. (A) One-step RT-PCR was conducted using Kit I with the indicated primer sets. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 35 (lanes 1, 3, 5 and 7) or 45 cycles (lanes 2, 4, 6 and 8) of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. Lanes 1, 2, 5 and 6: one-step RT-PCR with carrier RNA; Lanes 3, 4, 7 and 8: one-step RT-PCR without carrier RNA. Each reaction was carried out in duplicate. (B) One-step RT-PCR was conducted using Kit T (left panel) and Kit P (right panel) with primers 419F and 1154R with or without carrier RNA. The RT-PCR conditions using Kit T were as follows: reverse transcription at 50°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 30 s, 57°C for 30 s, and 72°C for 1 minute; and a final extension at 72°C for 10 minutes. The RT-PCR conditions using Kit P were as follows: reverse transcription at 45°C for 45 minutes; activation at 95°C for 2 minutes; 45 cycles of the following steps: 95°C for 30 s, 57°C for 30 s, and 70°C for 45 s; and a final extension at 70°C for 5 minutes. (C) One-step RT-PCR was conducted with primers GAG-I-F and GAG-I-R using Kit I with or without RNaseA. Carrier RNA was not added to the reaction mixtures. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. (D) One-step RT-PCR was conducted using Kit I to amplify env region of the contaminants. One-step RT-PCR was carried out using two primer sets p-env1f and p-env1r (lane 1), and p-env3f and p-env5r (lane 2). The RT-PCR conditions were the same as in Figure 1C with the exception of the number of PCR cycles (60 cycles instead of 45 cycles). M: DNA size marker.

Figure 2

One-step RT-PCR for identification of contaminants in Kit I and Platinum Taq. (A-C) One-step RT-PCR for identification of a contaminated component in Kit I. The experiments were conducted in two independent laboratories, IVR and JRC. In IVR, nucleic acids were extracted from 50 μl of the enzyme mix of the RT-PCR Kit I using an RNA purification column (QIAamp viral RNA mini kit [Cat. no. 52904] [QIAGEN]) and the presence of polytropic endogenous MLV was examined by using the RT-PCR Kit T (A) and Kit P (B). In JRC, nucleic acids were extracted from 75 μl of the enzyme mix of RT-PCR Kit I using an RNA/DNA purification column (PureLink™ Viral RNA/DNA Kit [Cat. no. 12280-050] [Invitrogen]), and the presence of polytropic endogenous MLV was examined using Kit Q (C). Five μl of test samples were examined with primers indicated below the corresponding lanes. The RT-PCR conditions for Kit T and Kit P were the same as in Figure 1B. The RT-PCR conditions for Kit Q were as follows: reverse transcription at 50°C for 30 minutes; activation at 95°C for 15 minutes; 45 cycles of the following steps: 94°C for 30 s, 57°C for 30 s, and 72°C for 1 minute; and a final extension at 72°C for 10 minutes. Lanes 1 and 5, DW; lanes 2 and 6, column-purified carrier RNA (carrier); lanes 3 and 7, column-purified nucleic acids from enzyme mix (enzyme) of the Kit I; lanes 4 and 8, 1 μl buffer of the Kit I plus 4 μl DW (buffer). (D) One-step RT-PCR for the detection of MLV RNA in Platinum Taq. Nucleic acids were extracted from 50 μl of the Platinum Taq using an RNA purification column (QIAamp viral RNA mini kit [QIAGEN]) and the presence of MLV RNA was examined by using the RT-PCR Kit P. Five μl of test samples were examined with primers indicated below the corresponding lanes. The RT-PCR condition was the same as in Figure 1B with the exception of the PCR cycles (60 cycles instead of 45 cycles). Abbreviation; DW: distilled water. M: DNA size marker.

Figure 3

Sequence alignments of a partial gag region of the contaminant in Kit I with a PmERV chr 7, XMRV strain VP62, and MLV-like sequences derived from CFS patients (CFS types 1 to 3). Origins of the sequences used for the alignment are described in the Findings. Sequence alignments were performed using GENETYX Win ver. 6 (GENETYX, Shibuya, Tokyo, Japan).

Figure 4

Sequence alignments of a partial env region of the contaminant in Kit I with a PmERV chr 7. Origins of the sequences used for the alignment are described in the Findings. Sequence alignments were performed using GENETYX Win ver. 6 (GENETYX, Shibuya, Tokyo, Japan).