An improved method for detection of replication-competent retrovirus in retrovirus vector products.
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.
Uchida
Eriko
E
National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. uchida@nihs.go.jp
Sato
Koei
K
Iwata
Akiko
A
Ishii-Watabe
Akiko
A
Mizuguchi
Hiroyuki
H
Hikata
Mikio
M
Murata
Mitsuhiro
M
Yamaguchi
Teruhide
T
Hayakawa
Takao
T
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