title: An improved method for detection of replication-competent retrovirus in retrovirus vector products.
abstract: Contamination by replication-competent retrovirus (RCR) is one of the most important safety issues of retrovirus vector products for gene therapy clinical research. To improve the sensitivity of RCR detection and to shorten the assay period, we have developed a novel RCR detection method (infectivity RT-PCR method) based on real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) in combination with virus infection and a novel virus concentration method using polyethyleneimine (PEI)-conjugated magnetic beads. In this method, permissive cells were infected with RCR samples, and amplified RCR in the culture supernatants was adsorbed by PEI-beads. Then RCR RNA extracted from PEI-beads was quantified by real-time RT-PCR. We demonstrated that 1 infectious unit (iu) of RCR spiked in 10(6) cfu/ml of vector products could be detected within 3 days, and the sensitivity for viral detection was increased 3- to 10-fold compared with the direct S+L- assay. By this method, the presence of retroviral vector interfered with RCR detection only slightly. In conclusion, infectivity RT-PCR conducted in conjunction with virus concentration using PEI-beads can detect RCR more sensitively and rapidly than the conventional infectivity assay.
authors.affiliation: National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. uchida@nihs.go.jp
authors.forename: Eriko
authors.initials: E
authors.lastname: Uchida
authors.forename: Koei
authors.initials: K
authors.lastname: Sato
authors.forename: Akiko
authors.initials: A
authors.lastname: Iwata
authors.forename: Akiko
authors.initials: A
authors.lastname: Ishii-Watabe
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Mizuguchi
authors.forename: Mikio
authors.initials: M
authors.lastname: Hikata
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Murata
authors.forename: Teruhide
authors.initials: T
authors.lastname: Yamaguchi
authors.forename: Takao
authors.initials: T
authors.lastname: Hayakawa
mesh_terms: D000818:Animals; D002415:Cats; D002460:Cell Line; D003257:Consumer Product Safety; D005822:Genetic Vectors / Q000032:analysis*; D006801:Humans; D051379:Mice; D008979:Moloney murine leukemia virus; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014412:Tumor Virus Infections / Q000517:prevention & control*; D014779:Virus Replication