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 (CT) 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.
Clem et al. Virology Journal 2007 4:65 doi:10.1186/1743-422X-4-65