DETECTION OF WILD-TYPE CONTAMINATION IN A RECOMBINANT ADENOVIRAL PREPARATION BY PCR

A rapid and sensitive method of detecting wild-type virus contaminatio n is needed for the preparation of recombinant adenoviruses for adenov iral vector applications in which purified vectors free of wild-type v irus are required for preclinical studies and clinical trials. In resp onse to this demand, we developed a PCR assay that uses two pairs of p rimers in the same reaction to detect adenoviral E1 DNA with co-amplif ication of E2B DNA as an internal control. Template DNA preparation wa s simplified and required only 365 mu L of culture medium of 293 cells that displayed a cytopathic effect following adenovirus infection. Ev aluation of the sensitivity of the assay demonstrated that it detected the E1 DNA in a reconstitution of one plaque-forming unit (pfu) of wi ld-type virus in 10(9) pfu of recombinant viruses. This method may be useful for quality control in the production of adenoviral vectors fre e of wild-type virus for gene therapy applications.