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.