Quantitation of viral DNA by real-time PCR applying duplex amplification, internal standardization, and two-color fluorescence detection

A real-time PCR method was developed to quantitate viral DNA that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve. Applied to human parvovirus B19 DNA, the linear range was from 10(2) to at least 5 x 10(6) copies per mi of sample. The coefficient of variation was 0.29 using a run control of 2,876 copies per mi, The method reduces the risk of false-negative results, yields high precision, and is applicable for other DNA targets.