Detection of wild-type contamination in a recombinant adenoviral preparation by PCR.
A rapid and sensitive method of detecting wild-type virus contamination is needed for the preparation of recombinant adenoviruses for adenoviral vector applications in which purified vectors free of wild-type virus are required for preclinical studies and clinical trials. In response to this demand, we developed a PCR assay that uses two pairs of primers in the same reaction to detect adenoviral E1 DNA with co-amplification of E2B DNA as an internal control. Template DNA preparation was simplified and required only 365 microL of culture medium of 293 cells that displayed a cytopathic effect following adenovirus infection. Evaluation of the sensitivity of the assay demonstrated that it detected the E1 DNA in a reconstruction of one plaque-forming unit (pfu) of wild-type virus in 10(9) pfu of recombinant viruses. This method may be useful for quality control in the production of adenoviral vectors free of wild-type virus for gene therapy applications.
Zhang
W W
WW
Department of Thoracic and Cardiovascular Surgery, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Koch
P E
PE
Roth
J A
JA
D000256:Adenoviridae / Q000235:genetics / Q000302:isolation & purification*; D017498:Adenovirus E1 Proteins / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D017498:Adenovirus E1 Proteins; D017931:DNA Primers; D004279:DNA, Viral