Figure 5.

Real-Time PCR Quantitation of Adenovirus Type 17 DNA Template. A. PCR-amplified genomic DNA from Adenovirus Type 17 was quantitated by spectrophotometry (OD 260 nm) and the genome equivalents/ml calculated based on Avogadro's number (6.02205 × 10²³/mol). Real-time PCR was conducted with Adenovirus Type 17 specific primers using 10⁹ to 10¹ genome equivalents/ml as template and then using 1 μl of the stock solution of Adenovirus Type 17 as template. A standard curve was produced based on the threshold cycle number of the tested concentrations of Adenovirus Type 17 DNA template and plotted compared to the threshold cycle number (C_T) of the stock solution of Adenovirus Type 17. B. The indicated genome equivalents of Adenovirus Type 17 were added to 1 ml of human plasma, treated with 220 nm filtration and DNAse/RNAse digestion. Total nucleic acids then were purified, amplified by random multiplex PCR (50 cycles) and sequenced as described in the Methods section.