Quantitative PCR Detection of EBV

The PCR primers for this assay were previously selected in the single-copy BALF-5 gene encoding the viral DNA polymerase [50 (link)]; the upstream and downstream primer sequences were 5′-CGGAAGCCCTCTGGACTTC-3′ and 5′-CCCTGTTTATCCGATGGAATG-3′, respectively, with a fluorogenic probe (5′-TGTACACGCACGAGAAATGCGCC-3′) with a sequence located between the PCR primers. Detectable DNA from EBV was identified by a real-time PCR assay as previously described [51 (link),52 (link)]. Briefly, DNA from whole blood samples collected in EDTA was extracted using Purogene blood core kit B (Qiagen, Minneapolis, MN, USA). The PCR reaction was performed using a mixture containing 1μL of DNA, 0.2 μM each primer, 0.1 μM fluorogenic probe, and 5 μL of TaqMan Master Mix (PE Applied Biosystems), and the PCR cycle was performed as follows: 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. The TaqMan Master Mix (PE Applied Biosystems) was used for all reactions. For all PCR analyses, water was used as negative control, and B958 and P3HR1 viral DNA were used as positive controls. The B958 and P3HR1 viral strains were kindly provided by Dr. Talita A. F. Monteiro (Federal University of Pará, PA, Brazil) and were described elsewhere [53 (link)]. Samples were defined as negative if the CT values exceeded 40 cycles.