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.