title: Caution in evaluation of removal of virus by filtration: Misinterpretation due to detection of viral genome fragments by PCR.
abstract: The testing of biological products at different stages of the manufacturing process currently involves quantitative polymerase chain reaction (Q-PCR)-based assays. Q-PCR techniques are able to detect not only the viral genome in viral particles but also fragments of degraded genome in samples. The ability of 15 and 19-nm filters to remove viruses was examined by conducting infectivity assays and Q-PCR assays using parvovirus B19 (B19), one of the smallest non-enveloped viruses. Although the filtered samples showed no infectivity, viral DNA was detected by Q-PCR. Interestingly, approximately 90% of the total viral genome in 15-nm filtrates had a detectable size of less than 0.5kb by the Q-PCR and as a consequence reduction factors were underestimated using Q-PCR. The reduction factors using Q-PCR might be underestimated due to the presence of a large amount of free B19 DNA which shows no infectivity in the tested filtrates. Therefore, the results of Q-PCR should be interpreted with caution. The careful design of primers is needed to eliminate amplification from fragments of viral DNA by Q-PCR.
authors.affiliation: Osaka Research Laboratory, Research and Development Division, Benesis Corporation, Osaka, Japan. Tsujikawa.Muneo@mc.mt-pharma.co.jp
authors.forename: Muneo
authors.initials: M
authors.lastname: Tsujikawa
authors.forename: Yuji
authors.initials: Y
authors.lastname: Ohkubo
authors.forename: Maki
authors.initials: M
authors.lastname: Masuda
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.forename: Kadue
authors.initials: K
authors.lastname: Takahashi
authors.forename: Yuko
authors.initials: Y
authors.lastname: Sasaki
authors.forename: Mikihiro
authors.initials: M
authors.lastname: Yunoki
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Ikuta
mesh_terms: D001688:Biological Products; D003666:Decontamination / Q000379:methods*; D005374:Filtration / Q000379:methods*; D016679:Genome, Viral; D016732:Parvovirus B19, Human / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D019562:Viral Load / Q000379:methods*
chemical_list: D001688:Biological Products