title: Detection of minute virus of mice using real time quantitative PCR in assessment of virus clearance during the purification of Mammalian cell substrate derived biotherapeutics.
abstract: A real time quantitative PCR assay has been developed for detecting minute virus of mice (MVM). This assay directly quantifies PCR product by monitoring the increase of fluorescence intensity emitted during enzymatic hydrolysis of an oligonucleotide probe labelled covalently with fluorescent reporting and quenching dyes via Taq polymerase 5'-->3' exonuclease activity. The quantity of MVM DNA molecules in the samples was determined using a known amount of MVM standard control DNA fragment cloned into a plasmid (pCR-MVM). We have demonstrated that MVM TaqMan PCR assay is approximately 1000-fold more sensitive than the microplate infectivity assay with the lowest detection limit of approximately one particle per reaction. The reliable detection range is within 100 to 10(9) molecules per reaction with high reproducibility. The intra assay variation is <2.5%, and the inter assays variation is <6.5% when samples contain >100 particles/assay. When we applied the TaqMan PCR to MVM clearance studies done by column chromatography or normal flow viral filtration, we found that the virus removal factors were similar to that of virus infectivity assay. It takes about a day to complete entire assay processes, thus, the TaqMan PCR assay is at least 10-fold faster than the infectivity assay. Therefore, we concluded that this fast, specific, sensitive, and robust assay could replace the infectivity assay for virus clearance evaluation.
authors.affiliation: Virology R&D Laboratory, Department of Cell Culture and Fermentation R&D, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Dejin
authors.initials: D
authors.lastname: Zhan
authors.forename: Margaret R
authors.initials: MR
authors.lastname: Roy
authors.forename: Christine
authors.initials: C
authors.lastname: Valera
authors.forename: Jesse
authors.initials: J
authors.lastname: Cardenas
authors.forename: Joann C
authors.initials: JC
authors.lastname: Vennari
authors.forename: Janice W
authors.initials: JW
authors.lastname: Chen
authors.forename: Shengjiang
authors.initials: S
authors.lastname: Liu
mesh_terms: D000818:Animals; D001483:Base Sequence; D001688:Biological Products / Q000302:isolation & purification; D002460:Cell Line; D002853:Chromatography, Liquid; D003588:Cytopathogenic Effect, Viral; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004340:Drug Contamination; D006801:Humans; D051379:Mice; D008915:Minute Virus of Mice / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D014462:Ultrafiltration; D014773:Virology / Q000379:methods
chemical_list: D001688:Biological Products; D017931:DNA Primers; D004279:DNA, Viral