Thermocycling was performed by denaturation for 3 min at 94°C followed by 45 cycles. Each cycle consisted of 1′ at 94°C, 1′ at 60°C, and 1′ at 72°C.
For Southern blot analysis, 20 μL of the PCR reaction was loaded onto and run in a 2% agarose gel for 3 h at 100 V. The gel was stained with ethidium bromide to visualize the PCR products, and then the DNA samples were transferred overnight in 0.4 M NaOH to a nylon membrane (Hybond N + , Amersham Pharmacia Biotech; Uppsala, Sweden). Hybridization was carried out using a SV40-specific P-end-labeled internal oligoprobe. Hybridization was performed overnight in 10 mL of hybridization solution containing the following: 5 X Denhardt’s solution, 0.5% sodium dodecyl sulfate, and 100 μg/mL of salmon sperm DNA at 52°C. Filters were washed at 52°C with a final stringency of 0.5 X sodium saline citrate, 0.1% sodium dodecyl sulfate and exposed to radiograph film at room temperature for < 30 min. Link
The DNA products obtained with the primers SV5 and SV6 were always sequenced to confirm their identity. The PCR product was purified using a commercially available kit (QIA-quick gel extraction kit; Qiagen; Santa Clarita, CA) according to the suggested protocol of the manufacturer. Both strands of the PCR product were then sent to a sequencing facility for final identification (Kimmel Cancer Institute, Jefferson University; Philadelphia, PA).
In the initial PCR screening of the DNA samples isolated from lymphomas, we used the set of primers SV5/SV6 that amplifies the RB-pocket binding domain of SV40 Tag. In our experience, this is a very sensitive set of primers for the detection of SV40 in human tumors. As indicated in Table 1 and shown in Figures 1 and 2, top, 3 of 29 lymphomas in the nonimmunocompromised group, 6 posttransplant lymphoproliferative disorders, and 2 AIDS lymphomas tested positive. All of the positive samples were of B-cell lineage.
Table 1—SV40-Positive Samples
Figure 1. Top, A and bottom, B: Southern blot of the PCR products obtained from 29 non-Hodgkin’s lymphoma DNA samples using the SV5/SV6 set of primers. Samples 11, 22, and 26 are positive. ( —) = H2O, negative control; ( + ) = SV40 DNA, positive control.
Figure 2. Top, A: Southern blot hybridization of the PCR products obtained from lymphoproliferative posttransplant disorder DNA samples (samples 1-23, 29, and 30) and from AIDS lymphoma DNA samples (samples 24-28) using the SV5/SV6 set of primers. Samples 4, 11, 12, 15, 24, 26, and 29 are positive. Prolonged exposure of this autoradiography revealed a faint reproducible positive signal in sample 17. Bottom, B:Southern blot hybridization of the PCR products obtained from DNA samples of selected immunocompromised patients using the R1/R2 set of primers. Samples 7, 15, and 26 are positive. (—) = H2O, negative control; (+) = SV40 DNA, positive control.