Quantitative PCR evaluation of parvovirus B19 removal via nanofiltration

•Fragmented B19 DNA prevents qPCR-based evaluation for B19 removal by nanofiltration.•qPCR for a long target sequence reduces amplification from fragmented B19 DNA.•Nuclease treatment allows qPCR for a long target sequence to quantify B19 particles.•qPCR-based accurate evaluation for B19 removal by nanofiltration is demonstrated. When human parvovirus B19 (B19) is removed from plasma-derived products by nanofiltration, non-infectious fragmented B19 DNA in filtrate prevents quantitative real time PCR (qPCR) from accurately evaluating reduction of the virus particles. To determine optimal target sequence length for detection of full-length B19 genome in the viral particles by qPCR, we analyzed 4 different sequences ranging from 372 to 1,980 bp and found that a 989 bp sequence shows a well-balanced performance for the sensitivity and the run time. Nuclease treatment of filtrates prior to qPCR is also expected to decrease the influence of the residual B19 DNA, but extremely high protein concentration of plasma-derived products in filtrates may result in incomplete digestion of the B19 DNA. In this context, however, our analysis showed that even when B19 genome is incompletely digested, qPCR for the 989 bp sequence successfully eliminates the influence of the B19 DNA. Finally, we verified that when B19-spiked plasma products are subjected to nanofiltration with the resulting filtrates treated with nuclease, qPCR for the 989 bp sequence accurately evaluates B19 removal. These results demonstrate that qPCR for the 989 bp sequence combined with nuclease treatment enables convenient and accurate evaluation of B19 removal by nanofiltration.