Supernatant rescue assay vs. polymerase chain reaction for detection of wild type adenovirus-contaminating recombinant adenovirus stocks.
The certification of recombinant adenoviruses prepared for clinical use requires the exclusion of contaminating, replication-competent adenovirus (wild type virus). Polymerase chain reaction (PCR)-based detection assays have been developed that detect the presence of viral sequences present only in wild type adenoviruses. As an alternative, this report describes a novel bioassay, designated the 'supernatant rescue assay', that detected minimal amounts of wild type virus mixed with high numbers of recombinant adenoviruses. This assay is based on the observation that minimal numbers of wild type adenovirus can be rescued from the supernatants of cells exposed to high multiplicities of infection (mois) of recombinant virus mixed with a known, minimal number of wild type virions. This assay was highly reproducible and routinely detected the presence of a single wild type virus mixed within 10(9) recombinant viruses. Under the experimental conditions employed, the supernatant rescue assay was significantly more sensitive than all three different PCR-detection assays.
Dion
L D
LD
UAB School of Medicine, AL, USA.
Fang
J
J
Garver
R I
RI
D000260:Adenoviruses, Human / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D001681:Biological Assay / Q000379:methods*; D002461:Cell Line, Transformed; D017077:Culture Media, Conditioned; D003588:Cytopathogenic Effect, Viral; D004279:DNA, Viral / Q000032:analysis; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D010948:Viral Plaque Assay
D017077:Culture Media, Conditioned; D004279:DNA, Viral