Detection of EBV lytic-cycle transcripts by RT-PCR amplification followed by nested PCR in RNA preparations from cell mixtures containing decreasing amounts of EBV-positive cells. RNA samples from a total of 2 × 106 cells were used as a template (U, undiluted). Negative controls (N) were performed with 2 × 106 EBV-negative Hela S3 cells. Dilutions are 10−1 to 10−6. (A) Quality control for B95-8 RNA preparation using histone 3.3 RT-PCR.41 Primer-probe combinations used for detection of BZLF1 (B), BRLF1 (C), BALF2 (D), and BcLF1 (E) are shown in the diagrams below the relevant transcripts. For BZLF1, BALF2, and BcLF1, RT-PCR and nested PCR were performed with B95-8 cells. For BRLF1 RNA detection where transcripts could not be obtained with the B95-8 cell line, we used PMA-treated P3HR1/16 cells. Nested PCR products yielding the predicted size (indicated at right) were further controlled by Southern hybridization with a specific probe.
